Multimodal activation of the ubiquitin ligase SCF by Nedd8 conjugation

Conjugation of ubiquitin-like protein Nedd8 to cullins (neddylation) is essential for the function of cullin-RING ubiquitin ligases (CRLs). Here, we show that neddylation stimulates CRL activity by multiple mechanisms. For the initiator ubiquitin, the major effect is to bridge the approximately 50 A gap between naked substrate and E2 approximately Ub bound to SCF. The gap between the acceptor lysine of ubiquitinated substrate and E2 approximately Ub is much smaller, and, consequentially, the impact of neddylation on transfer of subsequent ubiquitins by Cdc34 arises primarily from improved E2 recruitment and enhanced amide bond formation in the E2 active site. The combined effects of neddylation greatly enhance the probability that a substrate molecule acquires >or= 4 ubiquitins in a single encounter with a CRL. The surprisingly diverse effects of Nedd8 conjugation underscore the complexity of CRL regulation and suggest that modification of other ubiquitin ligases with ubiquitin or ubiquitin-like proteins may likewise have major functional consequences.     

Neddylation-induced conformational control regulates cullin RING ligase activity in vivo

Cullin RING ligases (CRLs) constitute the largest family of ubiquitin ligases with diverse cellular functions. Conjugation of the ubiquitin-like molecule Nedd8 to a conserved lysine residue on the cullin scaffold is essential for the activity of CRLs. Using structural studies and in vitro assays, it has been demonstrated that neddylation stimulates CRL activity through conformational rearrangement of the cullin C-terminal winged-helix B domain and Rbx1 RING subdomain from a closed architecture to an open and dynamic structure, thus promoting ubiquitin transfer onto the substrate. Here, we tested whether the proposed mechanism operates in vivo in intact cells and applies to other CRL family members. To inhibit cellular neddylation, we used a cell line with tetracycline-inducible expression of a dominant-negative form of the Nedd8 E2 enzyme or treatment of cells with the Nedd8 E1 inhibitor MLN4924. Using these cellular systems, we show that different mutants of Cul2 and Cul3 and of Rbx1 that confer increased Rbx1 flexibility mimic neddylation and rescue CRL activity in intact cells. Our findings indicate that in vivo neddylation functions by inducing conformational changes in the C-terminal domain of Cul2 and Cul3 that free the RING domain of Rbx1 and bridge the gap for ubiquitin transfer onto the substrate.     

Nedd8-modification of Cul1 is promoted by Roc1 as a Nedd8-E3 ligase and regulates its stability

SCF is a ubiquitin ligase and is composed of Skp1, Cul1, F-box protein, and Roc1. The catalytic site of the SCF is the Cul1/Roc1 complex and RING-finger protein Roc1. It was shown earlier that when Cul1 was co-expressed with Roc1 in Sf-9 cells in a baculovirus protein expression system, Cul1 was highly neddylated in the cell, suggesting that Roc1 may function as a Nedd8-E3 ligase. However, there is no direct evidence that Roc1 is a Nedd8-E3 in an in vitro enzyme system. Here we have shown that Roc1 binds to Ubc12, E2 for Nedd8, but not to Ubc9, E2 for SUMO-1 and Roc1 RING-finger mutant, H77A, did not bind to Ubc12. In in vitro neddylation system using purified Cul1/Roc1 complex expressed in bacteria, Roc1 promotes neddylation of Cul1. These results demonstrate that Roc1 functions as a Nedd8-E3 ligase toward Cul1. Furthermore, Roc1 and Cul1 were ubiquitinylated in a manner dependent on the neddylation of Cul1 in vitro. In addition, Cul1 was degraded through the ubiquitin-proteasome pathway, and a non-neddylated mutant Cul1, K720R, was more stable than wild-type in intact cells. Thus, neddylation of Cul1 might regulate SCF function negatively via degradation of Cul1/Roc1 complex.     

Dcn1 functions as a scaffold-type E3 ligase for cullin neddylation

Cullin-based E3 ubiquitin ligases are activated through modification of the cullin subunit with the ubiquitin-like protein Nedd8. Dcn1 regulates cullin neddylation and thus ubiquitin ligase activity. Here we describe the 1.9 A X-ray crystal structure of yeast Dcn1 encompassing an N-terminal ubiquitin-binding (UBA) domain and a C-terminal domain of unique architecture, which we termed PONY domain. A conserved surface on Dcn1 is required for direct binding to cullins and for neddylation. The reciprocal binding site for Dcn1 on Cdc53 is located approximately 18 A from the site of neddylation. Dcn1 does not require cysteine residues for catalytic function, and directly interacts with the Nedd8 E2 Ubc12 on a surface that overlaps with the E1-binding site. We show that Dcn1 is necessary and sufficient for cullin neddylation in a purified recombinant system. Taken together, these data demonstrate that Dcn1 is a scaffold-like E3 ligase for cullin neddylation.     

Characterization of cullin-based E3 ubiquitin ligases in intact mammalian cells--evidence for cullin dimerization

Cullin-based E3 ligases are a large family of ubiquitin ligases with diverse cellular functions. They are composed of one of six mammalian cullin homologues, the Ring finger containing protein Roc1/Rbx1 and cullin homologue-specific adapter and substrate recognition subunits. To be active, cullin-based ligases require the covalent modification of a conserved lysine residue in the cullin protein with the ubiquitin-like protein Nedd8. To characterize this family of E3 ligases in intact cells, we generated a cell line with tetracycline-inducible expression of a dominant-negative mutant of the Nedd8-conjugating enzyme Ubc12, a reported inhibitor of cullin neddylation. Using this cell line, we demonstrate that the substrate recognition subunit Skp2 and the adaptor protein Skp1 are subject to Ubc12-dependent autoubiquitination and degradation. In contrast, cullin protein stability is not regulated by neddylation in mammalian cells. We also provide evidence that Cul1 and Cul3, as well as their associated substrate recognition subunits Skp2 and Keap1, respectively, homooligomerize in intact cells, suggesting that cullin-based ligases are dimeric. Cul3, but not Cul1 homooligomerization is dependent on substrate recognition subunit dimer formation. As shown for other E3 ubiquitin ligases, dimerization may play a role in regulating the activity of cullin-based E3 ligases.     

Profiling the Cross Reactivity of Ubiquitin with the Nedd8 Activating Enzyme by Phage Display

The C-terminal peptides of ubiquitin (UB) and UB-like proteins (UBLs) play a key role in their recognition by the specific activating enzymes (E1s) to launch their transfer through the respective enzymatic cascades thus modifying cellular proteins. UB and Nedd8, a UBL regulating the activity of cullin-RING UB ligases, only differ by one residue at their C-termini; yet each has its specific E1 for the activation reaction. It has been reported recently that UAE can cross react with Nedd8 to enable its passage through the UB transfer cascade for protein neddylation. To elucidate differences in UB recognition by UAE and NAE, we carried out phage selection of a UB library with randomized C-terminal sequences based on the catalytic formation of UB∼NAE thioester conjugates. Our results confirmed the previous finding that residue 72 of UB plays a “gate-keeping” role in E1 selectivity. We also found that diverse sequences flanking residue 72 at the UB C-terminus can be accommodated by NAE for activation. Furthermore heptameric peptides derived from the C-terminal sequences of UB variants selected for NAE activation can function as mimics of Nedd8 to form thioester conjugates with NAE and the downstream E2 enzyme Ubc12 in the Nedd8 transfer cascade. Once the peptides are charged onto the cascade enzymes, the full-length Nedd8 protein is effectively blocked from passing through the cascade for the critical modification of cullin. We have thus identified a new class of inhibitors of protein neddylation based on the profiles of the UB C-terminal sequences recognized by NAE.     

Neddylation and deneddylation of CUL-3 is required to target MEI-1/Katanin for degradation at the meiosis-to-mitosis transition in C. elegans

BACKGROUND: 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. RESULTS: Here, we identified and characterized the COP9/signalosome in C. elegans and showed that 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. CONCLUSIONS: We conclude that both neddylation and deneddylation of CUL-3 is required for MEI-1 degradation and propose that cycles of CUL-3 neddylation and deneddylation are necessary for its ligase activity in vivo.     

Conjugation of Nedd8 to CUL1 enhances the ability of the ROC1-CUL1 complex to promote ubiquitin polymerization

The SCF-ROC1 ubiquitin-protein isopeptide ligase (E3) ubiquitin ligase complex targets the ubiquitination and subsequent degradation of protein substrates required for the regulation of cell cycle progression and signal transduction pathways. We have previously shown that ROC1-CUL1 is a core subassembly within the SCF-ROC1 complex, capable of supporting the polymerization of ubiquitin. This report describes that the CUL1 subunit of the bacterially expressed, unmodified ROC1-CUL1 complex is conjugated with Nedd8 at Lys-720 by HeLa cell extracts or by a purified Nedd8 conjugation system (consisting of APP-BP1/Uba3, Ubc12, and Nedd8). This covalent linkage of Nedd8 to CUL1 is both necessary and sufficient to markedly enhance the ability of the ROC1-CUL1 complex to promote ubiquitin polymerization. A mutation of Lys-720 to arginine in CUL1 eliminates the Nedd8 modification, abolishes the activation of the ROC1-CUL1 ubiquitin ligase complex, and significantly reduces the ability of SCF(HOS/beta)(-TRCP)-ROC1 to support the ubiquitination of phosphorylated IkappaBalpha. Thus, although regulation of the SCF-ROC1 action has been previously shown to preside at the level of recognition of a phosphorylated substrate, we demonstrate that Nedd8 is a novel regulator of the efficiency of polyubiquitin chain synthesis and, hence, promotes rapid turnover of protein substrates.     

Neddylation modification of the U3 snoRNA-binding protein RRP9 by Smurf1 promotes tumorigenesis

Neddylation is a posttranslational modification that attaches ubiquitin-like protein Nedd8 to protein targets via Nedd8-specific E1-E2-E3 enzymes and modulates many important biological processes. Nedd8 attaches to a lysine residue of a substrate, not for degradation, but for modulation of substrate activity. We previously identified the HECT-type ubiquitin ligase Smurf1, which controls diverse cellular processes, is activated by Nedd8 through covalent neddylation. Smurf1 functions as a thioester bond-type Nedd8 ligase to catalyze its own neddylation. Numerous ubiquitination substrates of Smurf1 have been identified, but the neddylation substrates of Smurf1 remain unknown. Here, we show that Smurf1 interacts with RRP9, a core component of the U3 snoRNP complex, which is involved in pre-rRNA processing. Our in vivo and in vitro neddylation modification assays show that RRP9 is conjugated with Nedd8. RRP9 neddylation is catalyzed by Smurf1 and removed by the NEDP1 deneddylase. We identified Lys221 as a major neddylation site on RRP9. Deficiency of RRP9 neddylation inhibits pre-rRNA processing and leads to downregulation of ribosomal biogenesis. Consequently, functional studies suggest that ectopic expression of RRP9 promotes tumor cell proliferation, colony formation, and cell migration, whereas unneddylated RRP9, K221R mutant has no such effect. Furthermore, in human colorectal cancer, elevated expression of RRP9 and Smurf1 correlates with cancer progression. These results reveal that Smurf1 plays a multifaceted role in pre-rRNA processing by catalyzing RRP9 neddylation and shed new light on the oncogenic role of RRP9.     

Regulation of cullin-based ubiquitin ligases by the Nedd8/RUB ubiquitin-like proteins

The expression of the ubiquitin related protein Nedd8/RUB is essential for growth in most organisms. Nedd8/RUB has been shown to modify the cullin subunit of culling-based ubiquitin protein ligases (E3). Neddylation acts to regulate the function of these E3s and organisms with lesions in the neddylation process exhibit severe growth defects. In this review we describe the proteins that participate in neddylation and discuss a model for Nedd8/RUB regulation of ubiquitin ligase function.     

Conjugation to Nedd8 instigates ubiquitylation and down-regulation of activated receptor tyrosine kinases

When appended to the epidermal growth factor receptor (EGFR), ubiquitin serves as a sorting signal for lysosomal degradation. Here we demonstrate that the ubiquitin ligase of EGFR, namely c-Cbl, also mediates receptor modification with the ubiquitin-like molecule Nedd8. EGF stimulates receptor neddylation, which enhances subsequent ubiquitylation, as well as sorting of EGFR for degradation. Multiple lysine residues, located within the tyrosine kinase domain of EGFR, serve as attachment sites for Nedd8. A set of clathrin coat-associated binders of ubiquitin also bind Nedd8, but they undergo ubiquitylation, not neddylation. We discuss the emerging versatility of the concerted action of ubiquitylation and neddylation in the process that desensitizes growth factor-activated receptor tyrosine kinases.     

SCCRO (DCUN1D1) promotes nuclear translocation and assembly of the neddylation E3 complex

SCCRO/DCUN1D1/DCN1 (squamous cell carcinoma-related oncogene/defective in cullin neddylation 1 domain containing 1/defective in cullin neddylation) serves as an accessory E3 in neddylation by binding to cullin and Ubc12 to allow efficient transfer of Nedd8. In this work we show that SCCRO has broader, pleiotropic effects that are essential for cullin neddylation in vivo. Reduced primary nuclear localization of Cul1 accompanying decreased neddylation and proliferation in SCCRO(-/-) mouse embryonic fibroblasts led us to investigate whether compartmentalization plays a regulatory role. Decreased nuclear localization, neddylation, and defective proliferation in SCCRO(-/-) mouse embryonic fibroblasts were rescued by transgenic expression of SCCRO. Expression of reciprocal SCCRO and Cul1-binding mutants confirmed the requirement for SCCRO in nuclear translocation and neddylation of cullins in vivo. Nuclear translocation of Cul1 by tagging with a nuclear localization sequence allowed neddylation independent of SCCRO, but at a lower level. We found that in the nucleus, SCCRO enhances recruitment of Ubc12 to Cul1 to promote neddylation. These findings suggest that SCCRO has an essential role in neddylation in vivo involving nuclear localization of neddylation components and recruitment and proper positioning of Ubc12.     

Inhibition of cullin RING ligases by cycle inhibiting factor: evidence for interference with Nedd8-induced conformational control

Cycle inhibiting factor (Cif) is produced by pathogenic intracellular bacteria and injected into the host cells via a type III secretion system. Cif is known to interfere with the eukaryotic cell cycle by inhibiting the function of cullin RING E3 ubiquitin ligases (CRLs). Cullin proteins form the scaffold protein of CRLs and are modified with the ubiquitin-like protein Nedd8, which exerts important conformational control required for CRL activity. Cif has recently been shown to catalyze the deamidation of Gln40 in Nedd8 to Glu. Here, we addressed how Nedd8 deamidation inhibits CRL activity. Our results indicate that Burkholderia pseudomallei Cif (also known as CHBP) inhibits the deconjugation of Nedd8 in vivo by inhibiting binding of the deneddylating COP9 signalosome (CSN) complex. We provide evidence that the reduced binding of CSN and the inhibition of CRL activity by Cif are due to interference with Nedd8-induced conformational control, which is dependent on the interaction between the Nedd8 hydrophobic patch and the cullin winged-helix B subdomain. Of note, mutation of Gln40 to Glu in ubiquitin, an additional target of Cif, inhibits the interaction between the hydrophobic surface of ubiquitin and the ubiquitin-binding protein p62/SQSTM1, showing conceptually that Cif activity can impair ubiquitin/ubiquitin-like protein non-covalent interactions. Our results also suggest that Cif may exert additional cellular effects by interfering with the association between ubiquitin and ubiquitin-binding proteins.     

TIP120A associates with unneddylated cullin 1 and regulates its neddylation

The cullin-containing E3 ubiquitin ligases play an important role in regulating the abundance of key proteins involved in cellular processes such as cell cycle and cytokine signaling. We recently identified TIP120A as a cullin-interacting protein and found that TIP120A functions as a negative regulator of a ubiquitin ligase by interfering with the binding of Skp1 and an F box protein to CUL1. Here we show that TIP120A binds to the unneddylated CUL1 but not the neddylated one. The association of TIP120A with CUL1 requires both the N-terminal stalk and the C-terminal globular domain of CUL1. TIP120A efficiently inhibits neddylation of CUL1 but does not affect substrate-independent ubiquitination by CUL1/Rbx1, implying that it blocks the access of Nedd8 to the conjugation site but does not interfere with the interaction of the ubiquitin-conjugating enzyme with Rbx1. Our data suggest that the association/dissociation of TIP120A coupled to neddylation/deneddylation of CUL1 may play an important role in assembly and disassembly of Skp1-Cdc53/cullin-F box ubiquitin ligases.     

Neddylation and deneddylation regulate Cul1 and Cul3 protein accumulation

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). Here we show 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. We further show 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. We propose 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.     

Rbx1 Flexible Linker Facilitates Cullin-RING Ligase Function Before Neddylation and After Deneddylation

In ubiquitination, cullin-RING E3 ubiquitin ligases (CRLs) assist in ubiquitin transfer from ubiquitin-conjugating enzyme E2 to the substrate. Neddylation, which involves NEDD8 transfer from E2 to E3-cullin, stimulates ubiquitination by inducing conformational change in CRLs. However, deneddylation, which removes NEDD8 from cullin, does not suppress ubiquitination in vivo, raising the question of how neddylation/deneddylation exerts its effects. Using molecular-dynamics simulations, we demonstrate that before neddylation occurs, the linker flexibility of Rbx1, a CRL component, leads to conformational changes in CRLs that allow neddylation and initiation of ubiquitination. These large NEDD8-induced conformational changes are retained after deneddylation, allowing both initiation of the ubiquitination process and ubiquitin chain elongation after deneddylation. Furthermore, mutation of lysine, the cullin residue to which NEDD8 covalently attaches, dramatically reduces CRL conformational changes, suggesting that the acceptor lysine allosterically regulates CRLs. Thus, our results imply that neddylation stimulates ubiquitination by CRL conformational control via lysine modification.     

Protection of cullin-RING E3 ligases by CSN-UBP12

Neddylation, a process that conjugates the ubiquitin-like polypeptide NEDD8 to cullin proteins, activates cullin-RING ubiquitin ligases (CRLs). Deneddylation, in which the COP9 signalosome (CSN) removes NEDD8 from cullins, inactivates CRLs. However, genetic studies of CSN function conclude that deneddylation also promotes CRL activity. It has been proposed that a cyclic transition through neddylation and deneddylation is required for the regulation of CRL activity in vivo. Recent discoveries suggest that an additional level of complexity exists, whereby CRL components are targets for degradation, mediated either by autocatalytic ubiquitination or by unknown mechanisms. Deneddylation by CSN and deubiquitylation by CSN-associated ubiquitin-specific protease 12 protect CRL components from cellular depletion, thus maintaining the physiological CRL activities.