By using a mRNA differential display technique to search for salicylate suppressible genes, a cDNA in human foreskin fibroblasts has been identified that shows sequence homology to the cullin/Cdc53 (CUL) family genes, especially CUL-3. The full-length human CUL-3 (Hs-CUL-3) cDNA has been cloned. It encodes a 768-amino acid polypeptide and has a predicted molecular weight of 88,939. The amino acid sequence of Hs-CUL-3 shows 46% homology to that of its Caenorhabditis elegans ortholog, Ce-CUL-3, and 27% and 23% to that of Hs-CUL-1 and Hs-CUL-2, respectively. Northern blot analysis showed that phorbol 12-myristate 13-acetate increases the expression of Hs-CUL-3 mRNA in a concentration- and time-dependent manner, and this increase is inhibited by sodium salicylate. Hs-CUL-3 is widely expressed in human tissues, and its expression in cultured COLO205 colon cancer cells is increased when compared with that in normal colon cells. It is likely that Hs-CUL-3 is involved in cell proliferation control (Du, 1998).
Programmed destruction of regulatory proteins through the ubiquitin-proteasome system is a widely used mechanism for controlling signalling pathways. Cullins are proteins that function as scaffolds for modular ubiquitin ligases typified by the SCF (Skp1-Cul1-F-box) complex. The substrate selectivity of these E3 ligases is dictated by a specificity module that binds cullins. In the SCF complex, this module is composed of Skp1, which binds directly to Cul1, and a member of the F-box family of proteins. F-box proteins bind Skp1 through the F-box motif and substrates by means of carboxy-terminal protein interaction domains. Similarly, Cul2 and Cul5 interact with BC-box-containing specificity factors through the Skp1-like protein elongin C. Cul3 is required for embryonic development in mammals and Caenorhabditis elegans but its specificity module is unknown. A large family of BTB-domain proteins has been identified as substrate-specific adaptors for C. elegans CUL-3. Biochemical studies using the BTB protein MEL-26 (Drosophila homolog: CG9924) and its genetic target MEI-1 (Drosophila homolog: katanin 60) indicate that BTB proteins merge the functional properties of Skp1 and F-box proteins into a single polypeptide (Xu, 2003).
Cullins (CULs) are subunits of a prominent class of RING ubiquitin ligases. Whereas the subunits and substrates of CUL1-associated SCF complexes and CUL2 ubiquitin ligases are well established, they are largely unknown for other cullin family members. S. pombe CUL3 (Pcu3p) forms a complex with the RING protein Pip1p and all three BTB/POZ domain proteins encoded in the fission yeast genome. The integrity of the BTB/POZ domain, which shows similarity to the cullin binding proteins SKP1 and elongin C, is required for this interaction. Whereas Btb1p and Btb2p are stable proteins, Btb3p is ubiquitylated and degraded in a Pcu3p-dependent manner. Btb3p degradation requires its binding to a conserved N-terminal region of Pcu3p that precisely maps to the equivalent SKP1/F box adaptor binding domain of CUL1. It is proposed that the BTB/POZ domain defines a recognition motif for the assembly of substrate-specific RING/cullin 3/BTB ubiquitin ligase complexes (Geyer, 2003).
These results identified BTB/POZ proteins as components of Pcu3p/Pip1p ubiquitin ligase complexes. Four pieces of evidence suggest that BTB/POZ domain proteins are functionally equivalent to the SKP1/F box adaptor dimers determining the substrate specificity of CUL1-associate SCF complexes: (1) all three BTB/POZ proteins present in the fission yeast genome interact with Pcu3p/Pip1p complexes; (2) BTB/POZ domains are structurally related to SKP1; (3) N-terminal residues invariably conserved in all CUL3 homologs, including Pcu3p, cluster in the same region of CUL1 that mediates its interaction with SKP1/F box adaptor dimers. Both the Btb3p/Pcu3p interaction and Pcu3p-dependent Btb3p degradation depend on the integrity of this conserved N-terminal region. (4) Btb3p is ubiquitylated in vitro in a Pcu3p-dependent manner, a finding reminiscent of CUL1-dependent ubiquitylation and degradation of F box proteins. Taken together, these findings strongly suggest that the BTB/POZ domain proteins ubiquitously present in eukaryotes define a family of substrate-specific adaptors for CUL3. Since fission yeast encodes three different BTB/POZ domain proteins, all of which interact with Pcu3p and Pip1p, it may form a minimum of three distinct RING/cullin 3/BTB complexes (Geyer, 2003).
The concentrations and functions of many cellular proteins are regulated by the ubiquitin pathway. Cullin family proteins bind with the RING-finger protein Roc1 to recruit the ubiquitin-conjugating enzyme (E2) to the ubiquitin ligase complex (E3). Cul1 and Cul7, but not other cullins, bind to an adaptor protein, Skp1. Cul1 associates with one of many F-box proteins through Skp1 to assemble various SCF-Roc1 E3 ligases that each selectively ubiquitinate one or more specific substrates. Cul3, but not other cullins, binds directly to multiple BTB domains through a conserved amino-terminal domain. In vitro, Cul3 promotes ubiquitination of Caenorhabditis elegans MEI-1, a katanin-like protein whose degradation requires the function of both Cul3 and BTB protein MEL-26. It is suggested that in vivo there exists a potentially large number of BCR3 (BTB-Cul3-Roc1) E3 ubiquitin ligases (Furukawa, 2003).
SCF (Skp1-Cullin-F-box) complexes are a major class of E3 ligases that are required to selectively target substrates for ubiquitin-dependent degradation by the 26S proteasome. Conjugation of the ubiquitin-like protein Nedd8 to the cullin subunit (neddylation) positively regulates activity of SCF complexes, most likely by increasing their affinity for the E2 conjugated to ubiquitin. The Nedd8 conjugation pathway is required in C. elegans embryos for the ubiquitin-mediated degradation of the microtubule-severing protein MEI-1/Katanin at the meiosis-to-mitosis transition. Genetic experiments suggest that this pathway controls the activity of a CUL-3-based E3 ligase. Counteracting the Nedd8 pathway, the COP9/signalosome has been shown to promote deneddylation of the cullin subunit. However, little is known about the role of neddylation and deneddylation for E3 ligase activity in vivo. The COP9/signalosome has been identified and characterized in C. elegans; it promotes deneddylation of CUL-3, a critical target of the Nedd8 conjugation pathway. As in other species, the C. elegans signalosome is a macromolecular complex containing at least six subunits that localizes in the nucleus and the cytoplasm. Reducing COP9/signalosome function by RNAi results in a failure to degrade MEI-1, leading to severe defects in microtubule-dependent processes during the first mitotic division. Intriguingly, reducing COP9/signalosome function suppresses a partial defect in the neddylation pathway; this suppression suggests that deneddylation and neddylation antagonize each other. It is concluded that both neddylation and deneddylation of CUL-3 is required for MEI-1 degradation and it is proposed that cycles of CUL-3 neddylation and deneddylation are necessary for its ligase activity in vivo (Pintard, 2003a).
Many biological processes, such as development and cell cycle progression are tightly controlled by selective ubiquitin-dependent degradation of key substrates. In this pathway, the E3-ligase recognizes the substrate and targets it for degradation by the 26S proteasome. The SCF (Skp1-Cul1-F-box) and ECS (Elongin C-Cul2-SOCS box) complexes are two well-defined cullin-based E3-ligases. The cullin subunits serve a scaffolding function and interact through their C terminus with the RING-finger-containing protein Hrt1/Roc1/Rbx1, and through their N terminus with Skp1 or Elongin C, respectively. In Caenorhabditis elegans, the ubiquitin-ligase activity of the CUL-3 complex is required for degradation of the microtubule-severing protein MEI-1/katanin at the meiosis-to-mitosis transition. However, the molecular composition of this cullin-based E3-ligase is not known. The BTB-containing protein MEL-26 has been identified as a component required for degradation of MEI-1 in vivo. Importantly, MEL-26 specifically interacts with CUL-3 and MEI-1 in vivo and in vitro, and displays properties of a substrate-specific adaptor. These results suggest that BTB-containing proteins may generally function as substrate-specific adaptors in Cul3-based E3-ubiquitin ligases (Pintard, 2003b).
Cullin family proteins organize ubiquitin ligase (E3) complexes to target numerous cellular proteins for proteasomal degradation. Neddylation, the process that conjugates the ubiquitin-like polypeptide Nedd8 to the conserved lysines of cullins, is essential for in vivo cullin-organized E3 activities. Deneddylation, which removes the Nedd8 moiety, requires the isopeptidase activity of the COP9 signalosome (CSN). This study shows that in cells deficient for CSN activity, cullin1 (Cul1) and cullin3 (Cul3) proteins are unstable, and that to preserve their normal cellular levels, CSN isopeptidase activity is required. It is further shown that neddylated Cul1 and Cul3 are unstable - as suggested by the evidence that Nedd8 promotes the instability of both cullins - and that the unneddylatable forms of cullins are stable. The protein stability of Nedd8 is also subject to CSN regulation and this regulation depends on its cullin-conjugating ability, suggesting that Nedd8-conjugated cullins are degraded en bloc. It is proposed that while Nedd8 promotes cullin activation through neddylation, neddylation also renders cullins unstable. Thus, CSN deneddylation recycles the unstable, neddylated cullins into stable, unneddylated ones, and promotes cullin-organized E3 activity in vivo (Wu, 2005).
Cyclin E is an unstable protein that is degraded in a ubiquitin- and proteasome- dependent pathway. Two factors stimulate cyclin E ubiquitination in vivo: when it is free of its CDK partner, and when it is phosphorylated on threonine 380. The first of these pathways was pursued by using a two-hybrid screen to identify proteins that could bind only to free cyclin E. This resulted in the isolation of human Cul-3, a member of the cullin family of E3 ubiquitin-protein ligases. Cul-3 is bound to cyclin E but not to cyclin E-Cdk2 complexes in mammalian cells, and overexpression of Cul-3 increases ubiquitination of cyclin E but not other cyclins. Conversely, deletion of the Cul-3 gene in mice causes increased accumulation of cyclin E protein, and has cell-type-specific effects on S-phase regulation. In the extraembryonic ectoderm, in which cells undergo a standard mitotic cycle, there is a greatly increased number of cells in S phase. In the trophectoderm, in which cells go through endocycles, there is a block to entry into S phase. The SCF pathway, which targets cyclins for ubiquitination on the basis of their phosphorylation state, and the Cul-3 pathway, which selects cyclin E for ubiquitination on the basis of its assembly into CDK complexes, may be complementary ways to control cyclin abundance (Singer, 1999).
DNA topoisomerase I (TOP1)-DNA covalent complexes are the initial lesions produced by antitumor camptothecins (CPTs). The TOP1-directed drugs stimulate degradation of TOP1 via the ubiquitin-proteasome pathway. Proteasome inhibition prevents degradation of DNA-bound TOP1 and sustains high levels of covalent complexes, thus enhancing CPT-induced cell death. Consistent with this, increased degradation of TOP1-DNA covalent complexes was seen in acquired CPT-resistant cells. The resistant cells showed elevated expressions of Cul3, a member of the cullin family of E3 ubiquitin ligases. The reduction in Cul3 expression by small interfering RNA decreased degradation of TOP1-DNA covalent complexes. Conversely, Cul3 overexpression by stable transfection promoted covalent complex degradation and reduced CPT-induced cell death without affecting basal TOP1 expression levels. These results indicate that Cul3, by promoting proteasomal degradation of TOP1-DNA covalent complexes, becomes an important regulator for cellular CPT sensitivity (Zhang, 2004).
Transcription factor Nrf2 is a major regulator of genes encoding phase 2 detoxifying enzymes and antioxidant stress proteins in response to electrophilic agents and oxidative stress. In the absence of such stimuli, Nrf2 is inactive owing to its cytoplasmic retention by Keap1 and rapid degradation through the proteasome system. The contribution of Keap1 to the rapid turnover of Nrf2 (half-life of less than 20 min) was examined, and it was found that a direct association between Keap1 and Nrf2 is required for Nrf2 degradation. In a series of domain function analyses of Keap1, it was found that both the BTB and intervening-region (IVR) domains are crucial for Nrf2 degradation, implying that these two domains act to recruit ubiquitin-proteasome factors. Indeed, Cullin 3 (Cul3), a subunit of the E3 ligase complex, was found to interact specifically with Keap1 in vivo. Keap1 associates with the N-terminal region of Cul3 through the IVR domain and promotes the ubiquitination of Nrf2 in cooperation with the Cul3-Roc1 complex. These results thus provide solid evidence that Keap1 functions as an adaptor of Cul3-based E3 ligase. Nrf2 and Keap1 are the first reported mammalian substrate and adaptor, respectively, of the Cul3-based E3 ligase system (Kobayashi, 2004).
The concentrations and functions of many eukaryotic proteins are regulated by the ubiquitin pathway, which consists of ubiquitin activation (E1), conjugation (E2), and ligation (E3). Cullins are a family of evolutionarily conserved proteins that assemble by far the largest family of E3 ligase complexes. Cullins, via a conserved C-terminal domain, bind with the RING finger protein Roc1 to recruit the catalytic function of E2. Via a distinct N-terminal domain, individual cullins bind to a protein motif present in multiple proteins to recruit specific substrates. Cullin 3 (Cul3), but not other cullins, binds directly with BTB domains to constitute a potentially large number of BTB-CUL3-ROC1 E3 ubiquitin ligases. The human BTB-Kelch protein Keap1, a negative regulator of the antioxidative transcription factor Nrf2, binds to CUL3 and Nrf2 via its BTB and Kelch domains, respectively. The KEAP1-CUL3-ROC1 complex promotes NRF2 ubiquitination in vitro; knocking down Keap1 or CUL3 by short interfering RNA results in NRF2 protein accumulation in vivo. It is suggested that Keap1 negatively regulates Nrf2 function in part by targeting Nrf2 for ubiquitination by the CUL3-ROC1 ligase and subsequent degradation by the proteasome. Blocking NRF2 degradation in cells expressing both KEAP1 and NRF2 by either inhibiting the proteasome activity or knocking down Cul3, results in NRF2 accumulation in the cytoplasm. These results may reconcile previously observed cytoplasmic sequestration of NRF2 by KEAP1 and suggest a possible regulatory step between KEAP1-NRF2 binding and NRF2 degradation (Furukawa, 2005).
Dishevelled is a conserved protein that interprets signals received by Frizzled receptors. Using a tandem-affinity purification strategy and mass spectrometry proteins have been identified associated with Dishevelled, including a Cullin-3 ubiquitin ligase complex containing the BTB protein Kelch-like 12 (KLHL12). This E3 ubiquitin ligase complex is recruited to Dishevelled in a Wnt-dependent manner that promotes its poly-ubiquitination and degradation. Functional analyses demonstrate that regulation of Dishevelled by this ubiquitin ligase antagonizes the Wnt-beta-catenin pathway in cultured cells, as well as in Xenopus and zebrafish embryos. Considered with evidence that the distinct Cullin-1 based SCF(beta-TrCP)complex regulates beta-catenin stability, these data on the stability of Dishevelled demonstrates that two distinct ubiquitin ligase complexes regulate the Wnt-beta-catenin pathway (Angers, 2006).
Faithful cell-cycle progression is tightly controlled by the ubiquitin-proteasome system. A human Cullin 3-based E3 ligase (Cul3) has been identified that is essential for mitotic division. In a complex with the substrate-specific adaptors KLHL9 and KLHL13, Cul3 is required for correct chromosome alignment in metaphase, proper midzone and midbody formation, and completion of cytokinesis. This Cul3-based E3 ligase removes components of the chromosomal passenger complex from mitotic chromosomes and allows their accumulation on the central spindle during anaphase. Aurora B directly binds to the substrate-recognition domain of KLHL9 and KLHL13 in vitro, and coimmunoprecipitates with the Cul3 complex during mitosis. Moreover, Aurora B is ubiquitylated in a Cul3-dependent manner in vivo, and by reconstituted Cul3/KLHL9/KLHL13 ligase in vitro. It is thus proposed that the Cul3/KLHL9/KLHL13 E3 ligase controls the dynamic behavior of Aurora B on mitotic chromosomes, and thereby coordinates faithful mitotic progression and completion of cytokinesis (Sumara, 2007).
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