SCCRO3 (DCUN1D3) Antagonizes the Neddylation and Oncogenic Activity of SCCRO (DCUN1D1)

The activity of cullin-RING type ubiquitination E3 ligases is regulated by neddylation, a process analogous to ubiquitination that culminates in covalent attachment of the ubiquitin-like protein Nedd8 to cullins. As a component of the E3 for neddylation, SCCRO/DCUN1D1 plays a key regulatory role in neddylation and, consequently, cullin-RING ligase activity. The essential contribution of SCCRO to neddylation is to promote nuclear translocation of the cullin-ROC1 complex. The presence of a myristoyl sequence in SCCRO3, one of four SCCRO paralogues present in humans that localizes to the membrane, raises questions about its function in neddylation. We found that although SCCRO3 binds to CAND1, cullins, and ROC1, it does not efficiently bind to Ubc12, promote cullin neddylation, or conform to the reaction processivity paradigms, suggesting that SCCRO3 does not have E3 activity. Expression of SCCRO3 inhibits SCCRO-promoted neddylation by sequestering cullins to the membrane, thereby blocking its nuclear translocation. Moreover, SCCRO3 inhibits SCCRO transforming activity. The inhibitory effects of SCCRO3 on SCCRO-promoted neddylation and transformation require both an intact myristoyl sequence and PONY domain, confirming that membrane localization and binding to cullins are required for in vivo functions. Taken together, our findings suggest that SCCRO3 functions as a tumor suppressor by antagonizing the neddylation activity of SCCRO.     

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.     

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.     

NEDD8 modification of CUL1 dissociates p120(CAND1), an inhibitor of CUL1-SKP1 binding and SCF ligases

Cullin proteins assemble a large number of RING E3 ubiquitin ligases and regulate various physiological processes. Covalent modification of cullins by the ubiquitin-like protein NEDD8 activates cullin ligases through an as yet undefined mechanism. We show here that p120(CAND1) selectively binds to unneddylated CUL1 and is dissociated by CUL1 neddylation. CAND1 formed a ternary complex with CUL1 and ROC1. CAND1 dissociated SKP1 from CUL1 and inhibited SCF ligase activity in vitro. Suppression of CAND1 in vivo increased the level of the CUL1-SKP1 complex. We suggest that by restricting SKP1-CUL1 interaction, CAND1 regulated the assembly of productive SCF ubiquitin ligases, allowing a common CUL1-ROC core to be utilized by a large number of SKP1-F box-substrate subcomplexes.     

Neddylation and CAND1 independently stimulate SCF ubiquitin ligase activity in Candida albicans

SCF (Skp1-cullin/Cdc53-F-box protein) ubiquitin ligases bind substrates via the variable F-box protein and, in conjunction with the RING domain protein Rbx1 and the ubiquitin-conjugating enzyme Ubc3/Cdc34, catalyze substrate ubiquitination. The cullin subunit can be modified covalently by conjugation of the ubiquitin-like protein Rub1/NEDD8 (neddylation) or bound noncovalently by the protein CAND1 (cullin-associated, neddylation-dissociated). Expression of the Candida albicans CAND1 gene homolog CaTIP120 in Saccharomyces cerevisiae is toxic only in the presence of CaCdc53, consistent with a specific interaction between CaTip120 and CaCdc53. To genetically analyze this system in C. albicans, we deleted the homologs of RUB1/NEDD8, TIP120/CAND1, and the deneddylase gene JAB1, and we also generated a temperature-sensitive allele of the essential CaCDC53 gene by knock-in site-directed mutagenesis. Deletion of CaRUB1 and CaTIP120 caused morphological, growth, and protein degradation phenotypes consistent with a reduction in SCF ubiquitin ligase activity. Furthermore, the double Carub1(-/-) Catip120(-/-) mutant was more defective in SCF activity than either individual deletion mutant. These results indicate that CAND1 stimulates SCF ubiquitin ligase activity and that it does so independently of neddylation. Our data do not support a role for CAND1 in the protection of either the F-box protein or cullin from degradation but are consistent with the suggested role of CAND1 in SCF complex remodeling.     

TRIP12 functions as an E3 ubiquitin ligase of APP-BP1

The NEDD8 pathway plays an essential role in various physiological processes, such as cell cycle progression and signal transduction. The conjugation of NEDD8 to target proteins is initiated by the NEDD8-activating enzyme composed of APP-BP1 and Uba3. In the present study, we show that APP-BP1 is degraded by ubiquitin-dependent proteolysis. To study biological functions of TRIP12, a HECT domain-containing E3 ubiquitin ligase, we used the yeast two-hybrid system and identified APP-BP1 as its binding partner. Immunoprecipitation analysis showed that TRIP12 specifically interacts with the APP-BP1 monomer but not with the APP-BP1/Uba3 heterodimer. Overexpression of TRIP12 enhanced the degradation of APP-BP1, whereas knockdown of TRIP12 stabilized it. In vitro ubiquitination assays revealed that TRIP12 functions as an E3 enzyme of APP-BP1 and additionally requires an E4 activity for polyubiquitination of APP-BP1. Moreover, neddylation of endogenous CUL1 was increased in TRIP12 knockdown cells, while complementation of the knockdown cells with TRIP12 lowered neddylated CUL1. Our data suggest that that TRIP12 promotes degradation of APP-BP1 by catalyzing its ubiquitination, which in turn modulates the neddylation pathway.     

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.     

Dynamics of cullin-RING ubiquitin ligase network revealed by systematic quantitative proteomics

Dynamic reorganization of signaling systems frequently accompanies pathway perturbations, yet quantitative studies of network remodeling by pathway stimuli are lacking. Here, we report the development of a quantitative proteomics platform centered on multiplex absolute quantification (AQUA) technology to elucidate the architecture of the cullin-RING ubiquitin ligase (CRL) network and to evaluate current models of dynamic CRL remodeling. Current models suggest that CRL complexes are controlled by cycles of CRL deneddylation and CAND1 binding. Contrary to expectations, acute CRL inhibition with MLN4924, an inhibitor of the NEDD8-activating enzyme, does not result in a global reorganization of the CRL network. Examination of CRL complex stoichiometry reveals that, independent of cullin neddylation, a large fraction of cullins are assembled with adaptor modules, whereas only a small fraction are associated with CAND1. These studies suggest an alternative model of CRL dynamicity where the abundance of adaptor modules, rather than cycles of neddylation and CAND1 binding, drives CRL network organization.     

The COP9 signalosome inhibits p27(kip1) degradation and impedes G1-S phase progression via deneddylation of SCF Cul1

The COP9 signalosome (CSN) is a conserved protein complex with homologies to the lid subcomplex of the 26S proteasome. It promotes cleavage of the Nedd8 conjugate (deneddylation) from the cullin component of SCF ubiquitin ligases. We provide evidence that cullin neddylation and deneddylation is highly dynamic, that its equilibrium can be effectively modulated by CSN, and that neddylation allows Cul1 to form larger protein complexes. CSN2 integrates into the CSN complex via its C-terminal region and its N-terminal half region is necessary for direct interaction with Cul1. The polyclonal antibodies against CSN2 but not other CSN subunits cause accumulation of neddylated Cul1/Cul2 in HeLa cell extract, indicating that CSN2 is essential in cullin deneddylation. Further, CSN inhibits ubiquitination and degradation of the cyclin-dependent kinase inhibitor p27(kip1) in vitro. Microinjection of the CSN complex impeded the G1 cells from entering the S phase. Moreover, anti-CSN2 antibodies negate the CSN-dependent p27 stabilization and the G1/S blockage, suggesting that these functions require the deneddylation activity. We conclude that CSN inhibits SCF ubiquitin ligase activity in targeting p27 proteolysis and negatively regulates cell cycle at the G1 phase by promoting deneddylation of Cul1.     

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.     

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.     

Cutting edge: bacterial modulation of epithelial signaling via changes in neddylation of cullin-1

The human enteric flora plays a significant role in intestinal health and disease. Certain enteric bacteria can inhibit the NF-kappaB pathway by blockade of IkappaB-alpha ubiquitination. IkappaB-alpha ubiquitination is catalyzed by the E3-SCF(betaTrCP) ubiquitin ligase, which is itself regulated via covalent modification of the cullin-1 subunit by the ubiquitin-like protein NEDD8. Neddylation is a biochemical event associated with diverse cellular processes related to cell signaling, however, physiological regulation of cullin neddylation has not been described in mammalian systems. We report that interaction of nonpathogenic bacteria with epithelial cells resulted in a rapid loss of neddylated Cul-1 and consequent repression of the NF-kappaB pathway. This observation may explain the ability of intestinal bacterial communities to influence diverse eukaryotic processes in general and inflammatory tolerance of the mammalian intestinal epithelia specifically.     

Preferential interaction of TIP120A with Cul1 that is not modified by NEDD8 and not associated with Skp1

The SCF complex, which consists of the invariable components Skp1, Cul1, and Rbx1 as well as a variable F-box protein, functions as an E3 ubiquitin ligase. The mechanism by which the activity of this complex is regulated, however, has been unclear. The application of tandem affinity purification has now resulted in the identification of a novel Cul1-binding protein: TATA-binding protein-interacting protein 120A (TIP120A, also called CAND1). Immunoprecipitation, immunoblot, and immunofluorescence analyses with mammalian cells revealed that TIP120A physically associates with Cul1 in the nucleus and that this interaction is mediated by a central region of Cul1 distinct from its binding sites for Skp1 and Rbx1. Furthermore, TIP120A was shown to interact selectively with Cul1 that is not modified by NEDD8. The Cul1-TIP120A complex does not include Skp1, raising the possibility that TIP120A competes with Skp1 for binding to Cul1. These observations thus suggest that TIP120A may function as a negative regulator of the SCF complex by binding to nonneddylated Cul1 and thereby preventing assembly of this ubiquitin ligase.     

CAND1 exchange factor promotes Keap1 integration into cullin 3-RING ubiquitin ligase during adipogenesis

Adipogenesis is governed by a plethora of regulatory proteins which are most commonly controlled by the ubiquitin proteasome system. Here, we show that the differentiation of LiSa-2 preadipocytes is associated with an increase of cullin-associated and neddylation-dissociated 1 (CAND1), COP9 signalosome (CSN), neddylated cullin 3 (Cul3) and the BTB protein Keap1. Silencing of CAND1 leads to a decrease and reduced integration of Keap1 into Cul3-RING ubiquitin ligases (CRL3) and to a retardation of adipogenesis. Transient transfection of LiSa-2 cells with CAND1 targeting miRNA148a also reduces Keap1 and slowed down adipogenesis of LiSa-2 cells. These results demonstrate for the first time that CAND1 acts as a BTB-protein exchange factor for CRL3 complexes. The specific increase of neddylated Cul3 might be explained by the recruitment of Cul3 or CRL3 in a membrane-bound location during adipogenesis. Together, the results show that during adipogenesis in LiSa-2 cells a CAND1-dependent remodeling and activation/neddylation of CRL3 complexes take place.     

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.     

The Cyclomodulin Cycle Inhibiting Factor (CIF) Alters Cullin Neddylation Dynamics

The bacterial effector protein cycle inhibiting factor (CIF) converts glutamine 40 of NEDD8 to glutamate (Q40E), causing cytopathic effects and inhibiting cell proliferation. Although these have been attributed to blocking the functions of cullin-RING ubiquitin ligases, how CIF modulates NEDD8-dependent signaling is unclear. Here we use conditional NEDD8-dependent yeast to explore the effects of CIF on cullin neddylation. Although CIF causes cullin deneddylation and the generation of free NEDD8 Q40E, inhibiting the COP9 signalosome (CSN) allows Q40E to form only on NEDD8 attached to cullins. In the presence of the CSN, NEDD8 Q40E is removed from cullins more rapidly than NEDD8, leading to a decrease in steady-state cullin neddylation. As NEDD8 Q40E is competent for cullin conjugation in the absence of functional CSN and with overexpression of the NEDD8 ligase Dcn1, our data are consistent with NEDD8 deamidation causing enhanced deneddylation of cullins by the CSN. This leads to a dramatic change in the extent of activated cullin-RING ubiquitin ligases.