Review

Oncogene (2004) 23, 1985–1997. doi:10.1038/sj.onc.1207414

Nedd8 on cullin: building an expressway to protein destruction

Zhen-Qiang Pan1, Alex Kentsis2, Dora C Dias1, Kosj Yamoah1 and Kenneth Wu1

  1. 1Derald H Ruttenberg Cancer Center, The Mount Sinai School of Medicine, New York, NY 10029-6574, USA
  2. 2Department of Physiology and Biophysics, The Mount Sinai School of Medicine, New York, NY 10029-6574, USA

Correspondence: Z-Q Pan, E-mail: zhen-qiang.pan@mssm.edu

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Abstract

This review summarizes recent advances concerning the Nedd8 regulatory pathway in four areas. One, substantial progress has been made in delineating the role of cullin family proteins, the only known substrates of the Nedd8 modification system. Cullins are molecular scaffolds responsible for assembling the ROC1/Rbx1 RING-based E3 ubiquitin ligases, of which several play a direct role in tumorigenesis. Two, a large body of work has helped elucidate the molecular details underlying the Nedd8 modification reaction, which results in covalent conjugation of a Nedd8 moiety onto a conserved cullin lysine residue. Three, studies using a variety of genetic model systems have established an essential role for Nedd8 in cell cycle control and in embryogenesis by upregulating the activities of cullin-based E3 ligases. In vitro experiments have revealed a direct role for Nedd8 in activating ubiquitination. Construction of a model of the ROC1/Rbx1-CUL1-Nedd8 structure suggests a mechanism by which the cullin-linked Nedd8 may assist the neighboring ROC1/Rbx1 in landing and positioning the E2 conjugating enzyme for the ubiquitin transfer reaction. Finally, increasing evidence indicates that removal of Nedd8 from its cullin targets, by the action of COP9 Signalosome and possibly other proteases, plays a significant role in the regulation of cullin-mediated proteolysis.

Keywords:

Nedd8, ubiquitin, proteolysis, cullins, E3 ligase

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Introduction

Nedd8/Rub1 is a small ubiquitin (Ub)-like protein, which was originally found to be conjugated to Cdc53, a cullin component of the SCF (Skp1-Cdc53/CUL1-F-box protein) E3 Ub ligase complex in Saccharomyces cerevisiae (Lammer et al., 1998; Liakopoulos et al., 1998). Initially, this discovery was greeted cautiously because mutant budding yeast cells lacking the Rub1/Nedd8 conjugation pathway are 'distressingly healthy' (Hochstrasser, 1998; Lammer et al., 1998). However, thanks to recent studies using both reconstituted ubiquitination systems and a variety of genetic model organisms, Nedd8 modification has now emerged as a regulatory pathway of fundamental importance for cell cycle control and for embryogenesis in metazoans. There are also substantial evidence suggesting an intimate link between Nedd8-activated proteolysis and tumorigenesis.

Cullins

To date, the only identified Nedd8 substrates are cullins, which are a family of structurally related proteins containing an evolutionarily conserved cullin domain (Kipreos et al., 1996; Yu et al., 1998; Zachariae et al., 1998). With the exception of APC2, each member of the cullin family is modified by Nedd8 (Hori et al., 1999; Gu, personal communication; Figure 1).

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Alignment of neddylation sites among cullin family members. Hori et al. (1999) have reported that hCUL1-5 are modified by Nedd8. Recently, the Gu laboratory found that both hCUL7 and Parc are substrates of Nedd8 as well (personal communication). The neddylation site (colored green) was experimentally determined for hCUL1, hCUL2 and scCUL8. The accession numbers of cullins are as follows: hCUL1 (NP_003583); hCUL2 (NP_003582); hCUL3 (NP_003581); hCUL4A (NP_003580); hCUL4B (NP_003579); hCUL5 (NP_003469); hCUL7 (NP_055595); scCUL8 (S56819) and Parc (NP_055904)

Full figure and legend (185K)

It is well established that several cullins function in Ub-dependent proteolysis, a process in which the 26S proteasome recognizes and subsequently degrades a target protein tagged with K48-linked polyubiquitin chains (Hershko and Ciechanover, 1998; Pickart, 2001). Signaling Ub chains are generated and attached to the substrate through the ubiquitination reaction typically mediated by an E3 Ub ligase. The E3 recognizes the substrate protein and also tethers an E2 Ub conjugating enzyme, thereby catalysing/promoting the transfer of Ub from the E2 to the target. There are two main classes of E3: ones containing a HECT domain and those possessing a RING finger motif. While HECT E3s utilize a conserved catalytic cysteine residue forming a thiol-ester intermediate with Ub, RING E3s recruit the E2 via the RING domain and promote the transfer of Ub from the E2 to a bound substrate.

Cullins, together with their RING finger partner ROC1/Rbx1 (also named Hrt1) (Kamura et al., 1999b; Ohta et al., 1999; Seol et al., 1999; Skowyra et al., 1999; Tan et al., 1999), define the largest subfamily of RING-based E3s. The interaction between a cullin and ROC1/Rbx1 assembles a core complex that supports the synthesis of polyubiquitin chains and was, therefore, initially defined as a core Ub ligase (Tan et al., 1999). Subsequent studies demonstrated that ROC1/Rbx1 alone can promote Ub ligation (Furukawa et al., 2002), albeit less efficiently (Pan, unpublished results). While the biological role and biochemical properties of cullins are summarized in Table 1, selective features of cullins are reviewed below with emphasis on recent advances and significance in cancer.


A prototype member of the cullin family, CUL1 is a subunit of the SCF E3 Ub ligase complex. The molecular action of CUL1 has been characterized both biochemically (Patton et al., 1998; Furukawa et al., 2000; Wirbelauer et al., 2000; Wu et al., 2000b) and structurally (Zheng N et al., 2002). Utilizing its distinct N- and C-terminal regions, CUL1 binds to the Skp1–F-box protein complex and to ROC1/Rbx1 (via the cullin domain), respectively. In this manner, CUL1 places an F-box protein (member of a large family of substrate-targeting molecules) within the proximity of ROC1/Rbx1, which recruits an E2 conjugating enzyme (Seol et al., 1999; Skowyra et al., 1999; Chen et al., 2000). Consequently, a substrate, once bound to the F-box protein, is positioned optimally for accepting an Ub moiety in an E2-catalysed transfer reaction. These findings have led to the hypothesis that cullins are scaffold proteins, responsible for assembling both a substrate-targeting module and a ROC1/Rbx1 RING-based Ub core ligase. The CUL1-mediated SCF pathway plays a significant role in cancer biology. For instance, defective regulation of the stability of the SCFbeta-TrCP substrate beta-catenin is evidently associated with tumor development (Morin, 1999).

CUL2 is a scaffold protein responsible for assembling the pVHL E3 Ub ligase complex containing the von Hipple–Lindau (pVHL) tumor suppressor protein, the elongin C/B dimer and ROC1/Rbx1 (reviewed in Conaway et al., 2002). In this assembly the N-terminus of CUL2 binds elongin C/B, a Skp1-like adaptor that interacts with the SOCS-box containing protein pVHL. This allows pVHL to target hypoxia inducible factor (HIF)-alpha for ubiquitination. Mutational inactivation of pVHL causes a predisposition to a variety of tumors, including clear cell renal carcinomas, cerebellar hemangioblastomas and hemangiomas, as well as pheochromocytomas. By targeting HIF-alpha for degradation, pVHL exerts its tumor suppressor function to negatively regulate hypoxia-inducible mRNAs encoding for vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT-1). HIF-alpha is rapidly degraded under normoxic conditions, but is stabilized by hypoxia (Salceda and Caro, 1997), through a prolyl hydroxylation-based regulatory mechanism (Ivan et al., 2001; Jaakkola et al., 2001). It turns out that the interaction with pVHL requires the hydroxylation of HIF-1alpha at proline residue 564 by the EGL-9/PHD family of prolyl hydroxylases (Epstein et al., 2001). These remarkable findings underscore a fundamental advance in cellular physiology, highlighting how changes in O2 concentration lead to altered gene expression, and defective regulation of this process by mutational inactivation of pVHL results in cancer.

The CUL3 E3 ligase appears to contain three subunits that include CUL3, ROC1/Rbx1 and a member of the BTB protein family (Furukawa et al., 2003; Geyer et al., 2003; Pintard et al., 2003b; Xu et al., 2003). While the BTB protein resembles the F-box protein in targeting substrates, it appears to utilize the BTB domain for tethering to CUL3 directly. There is substantial evidence suggesting that the BTB protein MEL-26 targets MEL-1 for degradation, an event important for transition from meiosis to mitosis in C. elegans (Pintard et al., 2003b).

Components of the CUL4A E3 Ub ligase complex were recently identified, exhibiting several attractive properties that may be critical to the regulation of both global nucleotide excision repair and transcription-coupled DNA repair (Groisman et al., 2003). CUL4A assembles a complex containing ROC1/Rbx1, DDB1 (UV-damaged DNA binding protein 1) and a WD40 repeat-containing protein, DDB2 or CSA (Cockayne syndrome gene product A). It can be speculated that DDB1 acts as a adaptor tethering DDB2 or CSA, which may resemble an F-box protein or pVHL to target a cognate substrate. Secondly, the core CUL4A ligase components (CUL4A, ROC1/Rbx1, DDB1 and DDB2 or CSA) formed a complex with the COP9 Signalosome (CSN; see below for details). Intriguingly, in an initial response to UV, the DDB2–DDB1–CUL4A–ROC1/Rbx1 complex was freed from the CSN and became associated with chromatin. Subsequently, the CSN rejoined the CUL4A–chromatin complex. However, UV treatment was found to cause the association between phosphorylated RNA polymerase II and the CSA–DDB1–CUL4A–ROC1/Rbx1–CSN complex. Consistent with these observations, a complex containing spCUL4/Pcu4, DDB1 homologue Ddb1 and CSN was isolated in fission yeast (Liu et al., 2003).

Another recent advancement is the identification of the CUL7 E3 ligase complex. CUL7 is a large polypeptide containing both the cullin and the DOC signature domains. The DOC domain, originally found in the APC10/DOC1 subunit of the APC/C (anaphase-promoting complex/cyclosome) E3 Ub ligase complex (Kominami et al., 1998), plays a role in enhancing the substrate recognition capacity of the APC (Carroll and Morgan, 2002; Passmore et al., 2003). Remarkably, CUL7 assembles an SCF–ROC1-like E3 ligase complex comprising Skp1, CUL7, the F-box protein Fbx29 and ROC1/Rbx1 (Dias et al., 2002; Arai et al., 2003). However, unlike CUL1, CUL7 specifically interacts with Skp1filled circleFbx29, but not with Skp1 alone nor with Skp1–betaTrCP2 or Skp1–Skp2 (Dias et al., 2002). Thus, while CUL1 serves as a molecular bridge that likely connects a large number of F-box proteins to an E2 conjugating enzyme through ROC1/Rbx1, CUL7 functions as an alternative scaffold, at least for Fbx29, owing to its ability to selectively interact with the Skp1–Fbx29 complex.

Work on mouse CUL7 may shed critical insights with respect to its biological function. Mouse CUL7 (named p185 or p193) was initially discovered as a SV40 large T-antigen (T-ag)-interacting protein (Kohrman and Imperiale, 1992; Tsai et al., 2000). The T-ag was found to interact with mouse CUL7 and p53 in a mutually exclusive manner. Mouse CUL7 exhibited a potent proapoptotic activity when overexpressed in NIH 3T3 cells, and coexpression of T-ag inhibited the CUL7-mediated proapoptotic activity. Recently, work by Arai et al. (2003) showed that deletion of the mouse CUL7 gene resulted in neonatal death, presumably due to abnormal vascular development. In addition, cul7-/- mouse embryonic fibroblasts exhibited slow growth. Taken together, these studies suggest roles for CUL7 in vascular development, cell proliferation and apoptosis.

Lastly, recent studies have identified intriguing functions associated with Parc, the largest cullin (270 kDa) of the family (Nikolaev et al., 2003). While nearly 70% of Parc shares extensive homology with CUL7, it possesses an additional C-terminal RING-IBR-RING domain resembling that of the Parkin E3 ligase. Parc binds at the N-terminus to p53 (Nikolaev et al., 2003). (And, CUL7 interacts with p53 as well, consistent with the high degree of conservation between the N-terminal domains of the two proteins; Dias and Pan, unpublished results.) Interestingly, forced expression of Parc led to cytoplasmic sequestration of p53. Of particular interest, abnormal cytoplasmic localization of p53 was found in a number of neuroblastoma cell lines, which contained high levels of Parc. Moreover, depletion of Parc by RNAi significantly sensitized these cells to apoptosis in response to DNA damage. Furthermore, it was recently observed that Parc coimmunoprecipitated Skp1 (Gu, personal communication), suggesting that it may resemble CUL7 in forming an SCF-like E3 ligase complex.

These recent advancements suggest that in addition to their scaffolding role for E3 ligase assembly, large cullins such as CUL7 and Parc mediate additional protein–protein interactions with cellular proteins including p53. Future studies will determine whether/how the interaction between p53 and Parc/CUL7 contributes to the regulation of cellular processes such as proliferation and apoptosis.

The Neddylation reaction

Nedd8 is covalently conjugated to a cullin protein through a process termed neddylation. Initially, Nedd8 forms, via its C-terminal glycine residue (Gly-76), a thiol-ester bond with a heterodimeric E1-activating enzyme called APP-BP1/Uba3. Following an exchange reaction, Nedd8 is then linked to a conserved cysteine on the Ubc12 E2 conjugating enzyme. Finally, neddylation is completed by formation of an isopeptide bond, linking the carboxyl-end of Nedd8 Gly-76 to the alt epsilon-amino group of a conserved cullin lysine residue. Previous publications have extensively reviewed the early work that led to the identification and characterization of Nedd8/Rub1, APP-BP1/Uba3 and Ubc12 (Hochstrasser, 1998; Yeh et al., 2000). Additionally, the catalytic properties as well as biological roles of APP-BP1/Uba3 and Ubc12 are summarized in Table 3.


Neddylation has been reconstituted in vitro with purified components (Kamura et al., 1999a; Wu et al., 2000a). The reaction included Nedd8, APP-BP1/Uba3, Ubc12 and a complex containing a cullin (CUL1/Cdc53 or CUL2) and ROC1/Rbx1. Incubation of these components in the presence of ATP yielded predominantly a conjugate comprised of the cullin protein and a single Nedd8 moiety, a characteristic initially observed in vivo for Cdc53 (CUL1 orthologue in S. cerevisiae; Lammer et al., 1998; Liakopoulos et al., 1998). Thus, in addition to its selective conjugation to cullin substrates, Nedd8 appears to differ from Ub by forming principally mononeddylated species.

Using site-directed mutagenesis, neddylation sites have been determined for human (h) CUL1 (K720; Read et al., 2000), hCUL2 (K689; Wada et al., 1999), S. pombe (sp) CUL3/Pcu3 (K729; Zhou et al., 2001), spCUL4/Pcu4 (K680; Osaka et al., 2000) and S. cerevisiae (sc) CUL8 (K791; Michel et al., 2003). It was initially noted by Wada et al. (1999) that the neddylation site is located within a conserved sequence, IVRIMKMR (Figure 1), although the significance of the amino acids flanking the lysine residue in neddylation has yet to be determined.

Previous work by Lammer et al. (1998) identified a Cdc53 C-terminal domain (residues 794–814; designated as CCD herein), located immediately downstream of the Nedd8 conjugation residue, as being required for neddylation in vivo. In support of this observation, deletion of the corresponding region in hCUL1, spanning residues 727–776, abolished the Nedd8 conjugation reaction in vitro (Yamoah and Pan, unpublished results). Also noted by Lammer et al. (1998) is the conservation of the CCD among cullin paralogues, suggesting a role for this motif in neddylation among all cullins. Additionally, Feldman et al. (1997) have shown that the C-terminal 60 residues of Cdc53 (CCD included) are dispensable for the ubiquitination of Sic1 by the reconstituted SCFCdc4. Together, these data suggest a specific role for the CCD in neddylation.

The X-ray crystallographic analysis of the CUL1–Rbx1/ROC1–Skp1–F-boxSkp2 complex by Zheng N. et al. (2002) revealed the structural information with respect to the neddylation site in hCUL1, K720CUL1. K720CUL1 is positioned at the rim of a 'cleft' formed by conserved residues from the CUL1 WH-B helix, four-helix bundle, as well as the ROC1/Rbx1 RING domain (Figure 2a; also see Figure 3c in Zheng N. et al., 2002). I715VRIM, the conserved residues preceding K720CUL1 (Figure 1), are a part of the H29 helix (Figure 2b). Additionally, the neddylation-essential CCD region starts in the middle of the H31 helix and contains both the S10 and S11 beta sheets (Figure 2b). Intriguingly, the CCD and residues V716, R717 and I718 in H29 are surface-exposed and located in close proximity to ROC1/Rbx1 (Figure 2a).

Figure 2.
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(a,b) Structure features of the human ROC1/Rbx1–hCUL1 complex in the vicinity of K720CUL1. Panel a is the surface representation of the ROC1/Rbx1–-hCUL1 complex in the vicinity of K720CUL1. The three-dimensional structure of Rbx1–hCUL1 was obtained from the PDB™ database (1LDK) at the Research Collaboratory for Structural Bioinformatics (RCSB) web site and visualized using the Swiss-PDBViewer software. The final image was rendered using the POV-ray 3.5 software. For simplicity, the truncated hCUL1, spanning amino-acid residues 330–776, was used in all figures. The surfaces of Rbx1/ROC1, hCUL1, hCUL1 CCD and the neddylation residue K720CUL1 are colored in red, green, orange and cyan, respectively. Conserved hCUL1 residues V716, R717, and I718, preceding K720CUL1, are colored in gray. The orientation of the hCUL1 N-terminus is indicated. Panel b is a close-up view of the structure of the hCUL1 C-terminal region (residues 700–776) with Rbx1/ROC1 bound. Rbx1/ROC1 and hCUL1 are colored in red and green, respectively. Helices and beta-sheets are numbered and underlined. Selected surface exposed amino-acid residues, K720CUL1 and the CCD are indicated. (c) A model for the neddylation reaction. CUL1 (green)-bound ROC1/Rbx1 (red) recruits Ubc12 (yellow), which may land on the CCD region as well. These complex interactions are thought to position the thiol ester-linked Nedd8 (blue) favorably for conjugation to K720CUL1 (purple-colored star). The orientation of the hCUL1 N-terminus is indicated

Full figure and legend (345K)

Figure 3.
Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

(a) Nedd8 charged surface residues reside on two distinct surfaces. The three-dimensional structure of Nedd8 was obtained from the PDB™ database (PDB ID #1NDD) at the Research Collaboratory for Structural Bioinformatics (RCSB) web site and visualized using the Swiss-PDBViewer software. The final image was rendered using the POV-ray 3.5 software. The side chains for Nedd8 amino acids K4, E12, E14, R25, E28, and E31 form patch 1 (colored yellow) and 2 (colored cyan), respectively. (b) A model of the ROC1/Rbx1–hCUL1–Nedd8 structure. The model was created by manually ligating hCUL1 K720 to Nedd8 G76 C-terminus through an isopeptide bond, by energy minimizing using CHARMM27, and by annealing after 0.5 ns molecular dynamics equilibration at 300 K in the presence of a distance-dependent dielectric screening function (alt epsilon=80) to mimic an aqueous environment. An average of 20 annealed structures is shown. hCUL1, ROC1/Rbx1, Nedd8 and Nedd8 patch 1 are colored blue, green, red and yellow, respectively. The orientation of the hCUL1 N-terminus is indicated. (c) A landing-and-positioning model for Nedd8. The attachment to hCUL1 (blue) at K720 places Nedd8 (red) in close proximity of ROC1/Rbx1 (green), creating a platform that may assist the landing of E2 (yellow) and Ub (cyan). The hCUL1-linked Nedd8 may also help position the bound E2approxSapproxUb for transferring Ub to the sequestered substrate (pink). The star (purple) represents a phosphorylated residue(s) on the substrate, which is typically a prerequisite for interaction with an F-box protein (gray) anchored to the N-terminal side of hCUL1 via the Skp1 adaptor (orange)

Full figure and legend (286K)

As revealed by in vitro studies, neddylation requires ROC1/Rbx1 as mutations disrupting the RING domain abolished the reaction (Kamura et al. 1999a). More recently, Dharmasiri et al. (2003) and Morimoto et al. (2003) demonstrated a direct interaction between ROC1/Rbx1 and Ubc12. On the basis of these findings, Dharmasiri et al. (2003) and Morimoto et al. (2003) proposed that ROC1/Rbx1 function as an E3 Nedd8-cullin isopeptide ligase, being capable of binding to both CUL1 and Ubc12. It can be postulated that neddylation is initiated by the binding of the CUL1-bound ROC1/Rbx1 to the Ubc12 E2 conjugating enzyme (Figure 2c). Residues within the CUL1 CCD region may engage in interactions with Ubc12 as well, perhaps to establish the optimal positioning of this E2 enzyme for the subsequent Nedd8 transfer reaction. Conceivably, owing to its ability to selectively interact with the cullin proteins, ROC1/Rbx1 ensures the specificity of the neddylation reaction, allowing only cullins to be modified by Nedd8.

Role of Nedd8 conjugation

It is now evident that the Nedd8 conjugation pathway is fundamentally important for cell cycle control and for embryogenesis in metazoans (see Table 2 for a summary). Studies in fission yeast have best exemplified the critical role for Nedd8 in cell growth (Osaka et al., 2000). In this organism, the Nedd8 conjugation system is necessary for cell viability as revealed by deletion analysis. Also, spCUL1/Pcu1 neddylation appears to be required for cell survival, because Pcu1K713R, a mutant deficient in Nedd8 modification, failed to rescue Pcu1-null cells. Furthermore, depletion of the Nedd8 conjugation activity resulted in stabilization of the SCF substrate Rum1. These studies suggest that optimal cellular SCF activity requires neddylation and that cell viability requires neddylated SCF.


In animals, the Nedd8 conjugation system is essential for embryonic development (Table 2). Furthermore, in both fruit fly and mouse, deficiency in the Nedd8 conjugation led to accumulation of SCF substrates such as beta-catenin/Armadillo and cyclin E (Tateishi et al., 2001; Ou et al, 2002). In Arabidopsis, Rce1 (Ubc12) mutation increased the level of SCFTIR1 substrate AXR2/IAA7 (Dharmasiri et al., 2003). Taken together, these results suggest that Nedd8 modification is required for proper SCF function in metazoans.

Additionally, it has been shown that IkappaBalpha, p27 and HIF-alpha were stabilized in the ts41 hamster cells at nonpermissive temperature, under which condition the Nedd8 E1-activating enzyme was inactivated (Ohh et al, 2002). Thus, the Nedd8 conjugation is also required for the function of CUL2, which, as described above, assembles the pVHL E3 that mediates the ubiquitination of HIF-alpha. Moreover, defective neddylation in C. elegans resulted in phenotypic defects similar to those observed with CUL3 depletion (Kurz et al., 2002), suggesting a role for Nedd8 in CUL3 function. Furthermore, in fission yeast, Pcu4K680R, a mutant form of spCUL4/Pcu4 deficient in the Nedd8 conjugation failed to complement growth defects in Pcu4-null cells (Osaka et al., 2000), underscoring a requirement for Nedd8 in CUL4 activity. All in all, these studies strongly support a role for Nedd8 in the upregulation of cullin family E3 Ub ligase activities.

How does Nedd8 act to activate the function of a cullin E3 ligase? Summarized below are key observations that led to several hypotheses concerning the mechanistic role of Nedd8.

Role for Nedd8 in assisting the landing and positioning of E2approxSapproxUb

Using in vitro systems, several studies have shown that Nedd8 activated the SCF-mediated ubiquitination of IkappaBalpha (Read et al., 2000; Kawakami et al., 2001) or p27 (Podust et al., 2000; Morimoto et al., 2000) through its conjugation to CUL1. Subsequently, it was observed that neddylation of CUL2 was also required for efficient ubiquitination of HIF-alpha by the pVHL E3 ligase (Ohh et al, 2002). A few additional points were revealed as well by these in vitro studies. Read et al. (2000) showed that neddylation did not affect the assembly of the SCFbeta-TrCP complex, nor did it alter the binding of this E3 to IkappaBalpha. Furthermore, highly purified components were used exclusively to show that SCFbeta-TrCP was more active than SCFbeta-TrCP containing CUL1K720R in the ubiquitination of IkappaBalpha (Kawakami et al., 2001). It is, therefore, unlikely that the observed activation is due to an additional factor. Together, these studies argue in favor of a direct role for Nedd8 in the activation of the E3 ligase function in ubiquitination.

In addressing a possible role for Nedd8 in the ROC1/Rbx1-promoted, E2-catalysed assembly of Ub chains, a purified reconstitution system was developed to allow efficient conjugation of Nedd8 to the ROC1/Rbx1-CUL1 complex expressed and isolated from bacteria (Wu et al., 2000a). (Hence, the CUL1 substrate used was devoid of any possible eucaryotic post-translational modification.) The results showed that the ROC1/Rbx1–CUL1–Nedd8 complex, repurified to remove excess neddylation agents, was significantly more active than the unmodified complex in its ability to support the assembly of multi-Ub chains catalysed by the Cdc34 E2 conjugating enzyme. Furthermore, in support of a role for Nedd8 in assisting E2 in substrate-dependent ubiquitination, it was shown that the Nedd8 conjugation directly increased the binding of SCFbetaTrCP to the Ubc4approxSapproxUb conjugate (Kawakami et al., 2001).

The activating function of Nedd8 is likely conferred by its unique structural organization, which mediates specific protein–protein interactions that lead to increased ubiquitination efficiency. As revealed by crystallographic studies (Whitby et al., 1998), six surface-exposed, charged residues (K4, E12, E14, R25, E28 and E31) are conserved among the various Nedd8 orthologs, but differ from Ub at the corresponding positions. As illustrated (Figure 3a), they are arranged in two surface patches that lie along each side of the Nedd8 molecule. Importantly, mutagenesis studies revealed that all six residues were required for the maximal activity of Nedd8 in promoting the ROC1/Rbx1-dependent assembly of Ub chains (Wu et al, 2002). In addition, the proper charges at amino-acid positions 14 and 25 were necessary for maintaining the Nedd8 activity, suggesting that Nedd8 mediates electrostatic interactions to assist ROC1/Rbx1 in the E2-catalysed polyubiquitin chain synthesis reaction.

To gain further insights into the mechanistic action of Nedd8, we have constructed a model for the ROC1/Rbx1–hCUL1–Nedd8 structure. As shown in Figure 3b, Nedd8 appears to dock into the conserved cleft in hCUL1, resulting from folding its isopeptide linked C-terminus into an alpha-helix, and from electrostatic interaction between its E31–E28–R25 surface (patch 2, Figure 3a) and hCUL1. Additionally, the conjugation appears to create contacts between Nedd8 and ROC1/Rbx1 as well. Notably, in this model, the E12–K4–E14 surface of Nedd8 (patch 1) remains solvent exposed (Figure 3b) and thus may play a critical role in establishing contacts with E2approxSapproxUb. Furthermore, our proposed model suggests that the Nedd8 conjugation may induce conformational changes within the ROC1/Rbx1–CUL1 complex. Such Nedd8-induced rearrangements may contribute to the interaction between ROC1/Rbx1 and E2approxSapproxUb.

We suggest a landing/positioning model to account for the molecular action of Nedd8 (Figure 3c). In this hypothesis, the hCUL1 K720-conjugated Nedd8 serves as a platform that assists ROC1/Rbx1 to land the incoming E2 and/or its thiol ester-linked Ub. Furthermore, the Nedd8 platform may help position the E2approxSapproxUb ester in a manner that favors the transfer of Ub to a substrate, which is sequestered by the F-box protein.

Nub1-mediated targeting of Nedd8 conjugates to the proteasome

Nub1 was identified as a Nedd8-interacting protein (Kito et al., 2001), which possesses an N-terminal Ub-like domain that interacted with S5a of the 19S proteasome activator (PA700) (Kamitani et al., 2001). Overexpression of Nub1 resulted in greater association of Nedd8 conjugates with GST-tagged S5a, suggesting that Nub1 may recruit Nedd8 and its conjugates to the proteasome for degradation (Kamitani et al., 2001). Future studies are required to demonstrate an Nub1-dependent association between the proteasome and a defined neddylated substrate.

Nedd8 conjugation reverses p120CAND1-mediated inhibition

p120CAND1 was found to selectively interact with unneddylated CUL1 and ROC1/Rbx1 (Liu et al., 2002; Zheng J. et al., 2002; Min et al., 2003; Oshikawa et al., 2003). Moreover, this interaction appeared to cause dissociation of Skp1 from CUL1, hence resulting in inhibition of SCF E3 ligase activity. Intriguingly, in vitro treatment of the p120CAND1 immunoprecipitates containing CUL1–ROC1/Rbx1 with the Nedd8 conjugation agents resulted in CUL1 modification and selective separation of neddylated CUL1 from p120CAND1, suggesting a role for neddylation in dissociating the CUL1–p120CAND1 complex. These observations further imply that neddylation may play a role in reversing the p120CAND1-mediated inhibition. This novel function of neddylation would be further substantiated if evidence could be provided to show that inactivation of the Nedd8 conjugation pathway in vivo (e.g., by the use of ts41 cells) would lead to increased association between CUL1 and p120CAND1 and to concomitant reduction of the CUL1–Skp1 interaction.

Influence of neddylation in cullin subcellular localization and stability

There has been no compelling evidence suggesting that Nedd8 modification influence the subcellular localization of cullins. It was shown that in Arabidopsis inactivation of Nedd8 conjugation did not alter nuclear localization of CUL1 (del Pozo et al., 2002). Additionally, a human CUL1 mutant, deficient in Nedd8 modification, was found to still localize to the nucleus in transfected cells (Furukawa et al., 2000). Thus, it appears that neddylation does not play a significant role in nuclear accumulation of CUL1.

It remains unclear whether neddylation plays a direct role in controlling the stability of cullins. Nedd8-null fruit fly mutants appear to accumulate CUL1 in third-instar eye disc (Ou et al., 2002). In Arabidopsis, it was observed that a defect in the Nedd8 conjugation increased the level of unmodified CUL1 (Dharmasiri et al., 2003). However, in Drosophila, neddylated CUL1 was accumulated at high levels in CSN5/Jab1 mutants (Doronkin et al., 2003). Clearly, neddylation-deficient mutants will be useful in addressing whether Nedd8 modification has a role in the turn over of cullin proteins.

Role of Nedd8 proteases

Recent studies have revealed the complexity of cellular activities that are involved in the removal of Nedd8 from its cullin targets (Table 3). Of particular interest is the identification of the CSN as a Nedd8 isopeptidase. The CSN is an eight-subunit complex that was originally identified as a suppressor of plant photomorphogenesis (Chamovitz and Glickman, 2002). In an attempt to isolate human SCF interacting proteins, abundant levels of all CSN subunits were found to copurify with hCUL1 that lacked its C-terminal region 692–752 (Lyapina et al., 2001). Strikingly, it was observed that deletion of a CSN subunit in S. pombe resulted in the accumulation of predominantly neddylated species of spCUL1/Pcu1, suggesting a critical role for the CSN in Nedd8 deconjugation in vivo. Furthermore, addition of purified CSN to yeast extracts containing neddylated spCUL1/Pcu1 resulted in removal of Nedd8 from its target, implicating a direct role for the CSN in promoting the cleavage reaction. In a subsequent study, Cope et al. (2002) have demonstrated that the JAMM metalloenzyme domain within the CSN-5/Jab1 subunit of the CSN was necessary for deneddylase activity, as mutations disrupting this motif accumulated neddylated cullins in S. pombe. Furthermore, the integrity of the putative JAMM catalytic domain was required for proper photoreceptor cell development in Drosophila. These results suggest that the CSN is a metalloprotease whose proteolytic activity is physiologically important. In support of this notion, recent studies using highly purified components showed that the CSN cleaved Nedd8 from hCUL1 catalytically (Wu et al., 2003). Additionally, in several organisms analysed (C. elegans, Drosophila and Arabidopsis), CSN activity is indispensable for deconjugation of Nedd8 from a number of tested cullins that include CUL1, CUL2 and CUL3 (Schwechheimer et al., 2001; Zhou et al., 2001; Yang et al., 2002; Doronkin et al., 2003; Pintard et al., 2003a). Surprisingly, requirement of the CSN for the deneddylation of spCUL4/Pcu4 might be different. In a study by Liu et al. (2003), deletion of CSN-1, but not CSN-4 or CSN-5/Jab1, appeared to accumulate neddylated spCUL4/Pcu4, suggesting that neither CSN-4 nor CSN-5/Jab1 was required for the deneddylation of this cullin protein. In summary, studies reported thus far seem to agree that the CSN contains an evolutionarily conserved deneddylase activity, which is likely required to regulate the neddylation status of all cullins. Detailed biochemical characterization is required to delineate the role for each CSN subunit in the assembly of the deneddylase activity.

Little is known about the mechanism by which the CSN cleaves Nedd8 from its cullin target. It is, however, conceivable that the CSN-mediated cleavage requires the interaction between the protease complex and a distinct structural motif that harbors the cullin neddylation site. In the modeled ROC1/Rbx1-CUL1-Nedd8 structure, the Nedd8 Gly76-K720CUL1 linkage remains surface-exposed (Figure 4). It can be speculated that the CSN may recognize ROC1/Rbx1 and/or a structural element within the C-terminus of CUL1. The active site of the CSN must then accommodate/position K720CUL1 as well as the Nedd8 Gly75–Gly76 motif, in a manner that is optimal for proteolytic cleavage. In support of this hypothesis, while CSN subunits 1 and 6 have been shown to interact with ROC1/Rbx1, CSN-2 appears to bind CUL1 in yeast two-hybrid assays (Lyapina et al., 2001; Schwechheimer et al., 2001; Pintard et al., 2003a). Additionally, purified CSN was found to bind to the ROC1/Rbx1–CUL1 complex in vitro (Yamoah and Pan, unpublished results).

Figure 4.
Figure 4 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Surface representation of the ROC1/Rbx1–hCUL1–Nedd8 complex in the vicinity of K720CUL1-Nedd8 Gly76. The ROC1/Rbx1–hCUL1–Nedd8 model was created as described in Figure 3. The surface of Rbx1/ROC1, hCUL1 and Nedd8 are colored in green, blue and red, respectively. hCUL1 K720 and Nedd8 Gly76 are in white and purple, respectively

Full figure and legend (165K)

Despite the identification of the CSN as a potent deneddylase in vivo and in vitro, the mechanistic role for this complex in cullin-mediated proteolysis remains to be elucidated. Using in vitro assays, Yang et al. (2002) have shown that the CSN inhibited p27 ubiquitination and degradation catalysed by HeLa cell cytosolic extracts. This inhibitory effect can be explained by the CSN deneddylase activity, which, by deconjugating Nedd8 from CUL1, is expected to reduce the SCF E3 ligase function. However, in Arabidopsis, reduced CSN expression levels resulted in stabilization of SCFTIR1 substrate PSIAA6, and had decreased auxin-response, similar to loss of function mutants of SCFTIR1 (Schwechheimer et al., 2001). In Drosophila, the CSN appears to play a major role in early oogenesis by promoting the degradation of cyclin E (Doronkin et al., 2003). Also, in C. elegans, RNAi-mediated reduction of CSN function accumulated neddylated CUL3 and stabilized MEI-1 (Pintard et al., 2003a). These results suggest that contrary to its inhibition of in vitro ubiquitination, the CSN is required for the degradation of cellular cullin E3 substrates.

It is now clear that the CSN has multiple cellular functions. It was found that removal of the N-terminus of Arabidopsis CSN1 resulted in photomorphogenic and growth defects without affecting deneddylase function (Wang et al., 2002). Additionally, fission yeast CSN was associated with the deubiquitylating enzyme Ubp12, which led to suppression of cullin Ub ligase activity (Zhou et al., 2003). There is an unexpected finding showing that CSN5/Jab1 mutants in Drosophila accumulated high levels of neddylated CUL1 in the cytoplasm (Doronkin et al., 2003), suggesting a role for the CSN in influencing the subcellular localization of cullins. Work from Liu et al. (2003) has provided strong evidence demonstrating a role for both CSN-1 and CSN-2 in activating ribonucleotide reductase (RNR). CSN-1 and CSN-2, via their cooperation with spCUL4/Pcu4, were found to induce the degradation of Spd1 (an inhibitor of RNR), thereby allowing the nuclear export of RNR. Intriguingly, in G2 cells after DNA damage, the CSN-1/2-mediated degradation of Spd1 also requires rad3- and chk1-dependent DNA damage checkpoint. Surprisingly, the Spd1 degradation pathway does not require CSN-4 or CSN-5/Jab1.

Given its eight-subunit composition and multiple associated biological activities, it is important to determine whether/how the CSN deneddylase activity contributes to the regulation of each specific function. It is presently unclear whether stabilization of cullin E3 substrates (PSIAA6, cyclin E and MEI-1), observed in organisms that lack a CSN subunit, or express reduced level of the CSN, can be attributed specifically to a loss of its deneddylase activity. This issue will be addressed satisfactorily with appropriate JAMM mutants that abolish only the CSN Nedd8 isopeptidase activity.

Cope and Deshaies (2003) have recently postulated a role for CSN in regulating cycles of SCF–ROC1 assembly. In their model, active SCF–ROC1 bound to substrate recruits CSN, which cleaves Nedd8 from CUL1. This allows the binding of p120CAND1 to CUL1, thereby displacing the Skp1–F-box protein heterodimer. Subsequent neddylation of CUL1 dissociates p120CAND1, resulting in reassociation of Skp1–F-box protein to yield an active SCF–ROC1 complex. This model suggests a significant role for neddylation in controlling the cycles of SCF–ROC1 assembly, which, conceivably, can be tested by inactivating the Nedd8 conjugation pathway in vivo (e.g., by the use of ts41 cells). In addition, cullin mutants deficient in neddylation would be expected to allow constitutive association with p120CAND1, hence exhibiting significant deficiency in SCF–ROC1 assembly.

Recently, three groups have reported studies on a novel Nedd8-speficifc protease called DEN1 (deneddylase 1; Gan-Erdene et al., 2003; Wu et al., 2003) or NEDP1 (Mendoza et al., 2003). DEN1 was previously annotated as SENP8 because it shares homology with the Ulp1/SENP cysteinyl SUMO deconjugating enzyme family (Li and Hochstrasser, 1999). Bacterially expressed human DEN1/NEDP1 was shown to bind to Nedd8 selectively and to hydrolyze C-terminal derivatives of Nedd8 efficiently and specifically (Mendoza et al, 2003; Gan-Erdene et al., 2003; Wu et al., 2003). These findings suggest a prominent role for DEN1 in processing the C-terminus of Nedd8 precursor (-G75G76GGLRQ), thereby generating the functional form of Nedd8 (-G75G76) for conjugation to cullins.

In addition, studies by both Wu et al. (2003) and Mendoza et al. (2003) have shown that DEN1/NEDP1 contains an isopeptidase activity capable of removing Nedd8 from its cullin targets. Incubation of high levels of GST–-NEDP1 with the CUL2–GST–Nedd8 conjugates, prepared in rabbit reticulocyte, resulted in cleavage of GST–Nedd8 from CUL2 (Mendoza et al., 2003). Coexpression of NEDP1, CUL4A and Nedd8 appears to decrease the levels of CUL4A-Nedd8 conjugates (Mendoza et al., 2003), implicating a role for NEDP1 in deconjugating cullin in vivo.

Yet, in a series of in vitro experiments carried out by Wu et al. (2003), DEN1 was shown to deconjugate CUL1-Nedd8 in a concentration-dependent manner. At a low concentration, DEN1 processed hyperneddylated CUL1 to yield a mononeddylated form, which presumably contains the K720CUL1-Nedd8 linkage. At elevated concentrations, DEN1 was able to complete the removal of Nedd8 from CUL1. Wu et al. (2003) postulated that the ability of a Nedd8 conjugate to be processed by DEN1 critically depends on the availability/accessibility of the Nedd8 moiety for interactions with the protease. While DEN1 can efficiently bind to, and cleave, the Nedd8 precursor, it may not readily recognize the K720CUL1-linked Nedd8, which appears to interact with both ROC1/Rbx1 and CUL1 (Figures 3b and 4). In contrast, DEN1 could conceivably recognize a Nedd8 moiety within hyperneddylated CUL1 that either is located at the distal end of a Nedd8 chain, or is conjugated to a non-K720 CUL1 lysine residue. It can thus be speculated that the main role for DEN1 as a Nedd8 isopeptidase is to maintain CUL1 (and other cullins) in mononeddylated forms, thereby reversing any hyperneddylation that might be disruptive of normal regulatory interactions for CUL1. Clearly, further studies are required to explore the physiological role for DEN1 in the deconjugation of Nedd8 from cullin targets.

Another likely role for DEN1 is in the salvage of adventitiously trapped derivatives of Nedd8. The C-terminal hydrolytic activity of DEN1 could be used to regenerate Nedd8 from adventitiously formed Nedd8 amides and thiol esters, in a manner analogous to the action of deubiqutinating enzymes (Pickart and Rose, 1985). Additionally, as it was observed that DEN1 deconjugated Nedd8 from Ubc12 in vitro (Wu and Pan, unpublished results), this protease may have a role in preventing the nonproductive autoneddylation of the E2 conjugating enzyme.

Conclusions and perspectives

As summarized in this review, a large body of work generated over the past 5 years has established an indispensable role for neddylation in the regulation of cell cycle and embryogenesis. Neddylation is a remarkably intricate biochemical process, ensuring that only the cullin proteins are modified by principally a single Nedd8 moiety. This sets a unique role for Nedd8 in the regulation of the cullin family E3 Ub ligases. Biochemical and structural analysis have suggested that a cullin-linked Nedd8 assists the neighboring ROC1/Rbx1 RING finger protein in assembling polyubiquitin chains, perhaps by stabilizing the RING–E2 interaction and helping position the E2 enzyme for the Ub transfer reaction. Recently, much excitement has been generated over the discovery of the CSN as a potent deneddylase activity in vivo and in vitro. Nedd8-activated, cullin-dependent proteolysis promotes unidirectional alteration of a divergent array of cellular processes. Defective regulation in these processes has been intimately linked to tumorigenesis.

Intriguingly, recent studies suggest that neddylation is a regulated cellular process. It was shown that in response to UV, the DDB2–DDB1–CUL4A–ROC1/Rbx1 complex, free of the CSN, became associated with chromatin (Groisman et al., 2003). Concomitantly, a substantial portion of CUL4A was found to exist in a neddylated form. Subsequently, however, the CSN joined the CUL4A ligase–chromatin complex, resulting in the conversion of neddylated CUL4A into its unmodified form. These findings imply that the Nedd8 modification status of a cullin may be determined predominantly by cellular deneddylase activities. Continued efforts to characterize the CSN and other Nedd8 proteases will promise a better understanding of how neddylation varies in response to developmental and environmental cues, thereby coordinating proteolytic events that are promoted by the cullin-based E3 Ub ligases.

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Acknowledgements

We thank A Capili for assistance in the preparation of Figure 2b; W Gu for communication of unpublished data and F Miller for a critical reading of the manuscript. AK is a graduate trainee in the laboratory of K Borden who is supported by NIH Grants CA 80728 and CA 88991. We apologize to those whose works on Nedd8 have not been cited here due to space restrictions. The studies carried out in the Pan laboratory were supported by Public Health Service Grants GM61051 and CA095634.

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