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RUB ubiquitin-like proteins
Seminars in Cell & Developmental Biology 15 (2004) 221–229
Regulation of cullin-based ubiquitin ligases by the Nedd8/RUB ubiquitin-like proteins Geraint Parry, Mark Estelle∗ Department of Biology, Indiana University, Myers Hall 150, 915 East Third Street, Bloomington, IN 47405, USA
Abstract 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. © 2004 Elsevier Ltd. All rights reserved. Keywords: Nedd8/RUB; Neddylation; Protein degradation; Cullin
1. Introduction In recent years several ubiquitin-like (Ubl) proteins have been identified in eukaryotes. These proteins are covalently attached to target proteins to facilitate a wide variety of cellular processes [1]. The RUB (related to ubiquitin) or Nedd8 (neural precursor cell-expressed developmentally downregulated 8) proteins are conserved throughout eukaryotes and exhibit approximately 55% identity to ubiquitin (Fig. 1a). Arabidopsis RUB1 has 83% identity with murine Nedd8 and 57% with ScRub1p (Fig. 1a) [2]. Nedd8/RUB molecules are post-translational modifiers of cullin proteins, subunits of E3 ubiquitin protein ligases. Despite intensive investigation in diverse species, no other substrate has been identified. A variety of studies have shown that the conjugation of Nedd8/RUB to cullin is important for E3 function. However, the precise function of Nedd8/RUB conjugation remains uncertain. In this article, we will review the pathways of Nedd8/RUB conjugation and deconjugation. Additionally, we will outline the cellular defects associated with loss of these activities and speculate on the role of Nedd8/RUB modification in SCF function. 2. The cullins Members of the cullin family of proteins have been identified in all eukaryotes. A recent phylogenetic analysis ∗ Corresponding
author. Tel.: +1-812-868-1216; fax: +1-812-855-6082. E-mail address: [email protected] (M. Estelle).
1084-9521/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcdb.2003.12.003
of the known eukaryotic cullins established five distinct clades [3]. Each clade contains a cullin protein from humans, Drosophila melanogaster and Caenorhabditis elegans. The Arabidopsis thaliana cullins CUL3A, CUL3B, and CUL4 group loosely with established clades. In contrast, Arabidopsis CUL1, the most highly studied plant cullin, lies together with the closely related Arabidopsis CUL2, outside of the five established cullin clades [3]. Cullins are subunits of at least four related ubiquitin protein ligases; the anaphase promoting complex/cyclosome (APC) [4,5], the SCF complex (SKP1 cullin F-box protein) [6], the VBC (VHL-elongin B-elongin C) [7,8], and a recently identified complex containing CUL3 and a member of the BTB family of proteins (the BTB complex) [9–11]. All cullins are modified by Nedd8/RUB except for the divergent APC subunit Apc2. Although this review will focus on the role of cullin in the SCF complex, it is important to note that Nedd8/RUB has an important role in the VBC and BTB complexes as well. So far the best understood cullin-based E3s are the SCFs. These complexes are comprised of CUL1, an F-box protein (FBP), SKP1, and RBX1 [6]. The function of CUL1 in this complex is to form a scaffold onto which the SKP1–FBP module and RBX1 bind [12,13]. The crucial role of CUL1 and SCFs in cellular regulation has been clearly shown through genetic studies in diverse species. In budding yeast, mutations in CDC53 (the CUL1 gene in this organism) result in cell cycle arrest at the G1/S transition [14]. Similar cell cycle defects as well as changes in mitotic chromosome condensation have been confirmed
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Fig. 1. Sequence alignments of the Nedd8/RUB proteins and the neddylation site on cullin proteins. (a) Nedd8/RUB proteins from Arabidopsis, human, mouse, Drosophila, Caenorhabditis elegans, Saccharomyces pombe, Saccharomyces cerevisiae and the ubiquitin protein from Arabidopsis. (b) Alignment of the C-terminal 200 amino acids of cullin proteins. The conserved Nedd8- and RBX1-binding sites are shown.
following removal of cullin activity in C. elegans [15]. In Arabidopsis, plants that lack CUL1 arrest at a very early stage in embryogenesis [16], as do CUL1-deficient mice [17].
3. Nedd8/RUB A single Nedd8 gene is present in humans, mice, flies, and nematodes. In contrast, Arabidopsis has three RUB genes
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(Fig. 1a). AtRUB1 and AtRUB2 differ at a single amino acid while AtRUB3 is 78% identical to AtRUB1. Unlike AtRUB3 and Nedd8/RUB orthologues in other species, AtRUB1 and AtRUB2 are synthesized as fusion proteins with ubiquitin [18]. Although the significance of this difference is not clear, it is conserved throughout angiosperms and therefore may be important. AtRUB1 and AtRUB2 are expressed in all organs in Arabidopsis while AtRUB3 is expressed only in flower buds and in the stem suggesting that AtRUB3 may have a unique role [2]. Nedd8 is expressed in all mouse tissues examined [19]. Immunohistochemistry of HA-Nedd8 transfected HeLa cells demonstrated that Nedd8 accumulates in the nucleus [20]. This is consistent with the localization of both CUL1 and enzymes in the Nedd8/RUB conjugation pathway to the nucleus in plant and animal species [16,20,21]. Independent crystallization of human Nedd8 and Arabidopsis RUB1 revealed that these proteins contain five -sheets, together with one significant ␣-helix, and two short ␣-helical regions [2,22]. This structure is extremely similar to ubiquitin and SUMO1. As discussed below and elsewhere in this volume, each Ubl is activated by a specific E1 enzyme. Several groups speculate that this specificity could be mediated in part by differences in surface electrostatic potential between Nedd8/RUB and ubiquitin [2,22,23]. In addition, a change from Arg72 in ubiquitin to Ala72 in Nedd8 (or Glu72 in SUMO) may contribute to specificity. This residue is known to be important for interaction with the E1. Further, the change from Arg to Ala corresponds to differences in the side chains of the E1 enzymes at the putative site of interaction. Therefore, Walden et al. [23] hypothesize that specific amino acids in each E1 can interact with the amino acid at residue 72 in their respective Ubl.
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conjugated to a conserved lysine near the C-terminus of the cullin (Fig. 1b). Although the identities of the Nedd8/RUB E1 and E2 have been known for several years, until recently it was not known if an additional E3 activity is required for attachment to the cullin. The main candidate for this role is the RBX1/Roc1 protein, which together with the cullin, is a core component of SCF, VBC, and BTB complexes [9,24–26]. RBX1/Roc1 binds to CUL1 in the C-terminal region of the protein near the site of Nedd8/RUB attachment (Fig. 1b) [13,27]. In vitro studies demonstrated that conjugation of Rub1p to Cdc53p is dependent on the presence of Rbx1p [28]. In more recent experiments, transgenic Arabidopsis that overexpressed RBX1 exhibited a large increase in the amount of modified CUL1, indicating that the RBX1 protein can facilitate attachment of RUB1, potentially as an E3 enzyme [26]. Further, Arabidopsis RBX1 directly binds the RUB E2 enzyme RCE1 suggesting that RBX1 is responsible for bringing the RUB E2, presumably carrying activated RUB, to the cullin protein [29]. Similarly, Morimoto et al. [30] have shown that human Roc1 binds to the Nedd8 E2, Ubc12. This group also demonstrated that a mutation in the RING domain of Roc1 prevents this interaction. Additionally, recombinant Roc1 promotes neddylation of CUL1 in vitro [30]. Unlike the case for most ubiquitin targets, cullin proteins are typically modified by a single Nedd8 molecule. However, several instances of hyperneddylation have been observed [31,32]. During the course of an in vitro neddylation experiment, Wu et al. [12] demonstrated that a small proportion of CUL1 was conjugated to two Nedd8 molecules, It is not clear if this represents a short Nedd8 chain, or Nedd8 attachment to different lysine residues.
5. Nedd8/RUB processing 4. The neddylation pathway Like ubiquitin conjugation, neddylation is accomplished by specific activating (E1) and conjugating (E2) enzymes. These enzymes are similar to those of the ubiquitin pathway except that the Nedd8/RUB E1, like that of other Ubls, consists of two subunits corresponding to N-terminal and C-terminal regions of the ubiquitin E1. The Nedd8/RUB E1 and E2 enzymes have been identified and characterized in many organisms and are listed in Table 1. Nedd8/RUB is
Like ubiquitin, conjugation of Nedd8/RUB requires a carboxyl-glycine [33]. Since all Nedd8/RUB molecules are synthesized with additional amino acids at their C-terminus, these need to be removed prior to attachment to target proteins. A yeast two-hybrid screen with Nedd8 as bait identified a human ubiquitin-C-terminal hydrolase (UHC-L3) [34]. UCH-L3 was shown to interact with both Nedd8 and ubiquitin in vitro but not with other Ubls. Further in vitro analysis proved that UCH-L3, but not the related enzyme
Table 1 Nedd8/RUB activating (E1) and conjugating (E2) enzymes Organism
Nedd8/RUB
E1
E2
Reference
Saccharomyces cerevisiae Saccharomyces pombe Arabidopsis thaliana
ScRub1p SpNedd8 AtRUB1
ENR2, Uba3b SpUba3 AXR1, ECR1
Ubc12 SpUbc12 RCE1
Caenorhabditis elegans Homo sapiens
Ned-8 Nedd8
Ula1, Uba3 APP-BP1, hUba3
Ubc12 hUbc12
Lammer et al. (1997), Liakopoulos et al. (1998) Osaka et al. (2000) Leyser et al. (1993), del Pozo et al. (1998), del Pozo and Estelle (1999), Dharmasiri et al. (2003) Jones and Candido (2000) Osaka et al. (1998), Gong and Yeh (1999)
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UCH-L1, could cleave the C-terminal region of Nedd8 to expose Gly76 [34]. Therefore, UCH-L3 may be involved in the maturation of both ubiquitin and Nedd8. Similarly, the Saccharomyces cerevisiae orthologue of UCH-L3, called Yuh1p, is required for Rub1p C-terminal cleavage and subsequent conjugation to Cdc53p [35]. In contrast to UCH-L3, a recently identified protein from mammalian cells called DEN1 (DENeddylase 1) selectively cleaves Nedd8 [36,60,61]. However, the biological roles of these two enzymes are not known at present.
6. CAND1 A novel protein that acts in the control of SCF function was recently independently discovered by four research groups. This protein is called CAND1 [37], p120CAND1 [38], or TIP120A [39–41]. TIP120A had been described earlier because of its ability to bind the TATA-binding protein [42,43]. In the context of the SCF, CAND1 forms a complex with CUL–RBX1, thus inhibiting the interaction between CUL1 and SKP1. Most revealing, CAND1 preferentially binds deneddylated CUL1 [37,38,40,41]. Further, neddylation of CUL1 promotes the dissociation of CAND1, thus permitting SCF formation [37,38]. Although the four groups agree on most aspects of the CUL1–CAND interaction, Oshikawa et al. [41] report that TIP120A is only able to interact with CUL1 whereas the other groups demonstrate CAND1 binding to other cullins [37–39]. The removal of the C-terminal 31 amino acids from CUL1 prevents CAND1 binding [37,40]. Because this region encompasses the neddylation site, CAND1 may be unable to bind Nedd8–CUL because of steric hindrance due to the presence of Nedd8 near the CAND1-binding site [40]. The converse does not appear to be true as Nedd8 attachment is possible in the presence of CAND1 in HeLa cell extracts [37] and with preformed CUL1–CAND1 complexes in vitro [38]. Additionally, two groups demonstrate that CAND1 binds to an N-terminal region of CUL1, an interaction that prevents SKP1–CUL1 complex formation [37,40]. On the other hand, Oshikawa et al. [41] report that the crucial region for CAND1 binding is the central portion of CUL1. CAND1 affects the neddylation process by binding cullin. In contrast, the recently identified ASPP2 (apoptosis stimulating protein of p53) inhibits the process at its primary step [44]. ASPP2 interacts with APP-BP1, a subunit of the Nedd8 E1. When ASPP2 is expressed in HeLa cells the proportion of unconjugated CUL1 increases suggesting that this protein may have a role in regulation of the Nedd8 pathway.
7. Function of the neddylation pathway The loss of individual cullins results in severe defects in growth and development in all species examined [15–17,45]. In addition, it is clear from experiments in which compo-
nents of the neddylation pathway are removed that cullin function is dependent on its neddylation state. With the exception of the yeast S. cerevisiae, defects in the Nedd8/RUB conjugation pathway result in dramatic phenotypes. In S. pombe, loss of SpNedd8, SpUba3 (E1 subunit), or SpUbc12 (E2) resulted in cell cycle arrest [45]. In C. elegans RNAi mediated reduction in the expression of ceNED-8 and ceUBC-12 results in arrested embryonic development or severe post-embryonic abnormalities [46]. In mice, a defect in cell division is thought to be responsible for the lethality of Uba3 homozygous mice [47]. In utero dissection revealed that the inner cell mass of the mutant embryo underwent premature apoptosis. The mutant trophoblastic cells were unable to enter S phase of the cell cycle [47]. The crucial role of the Nedd8 pathway in humans was demonstrated by overexpression of a dominant negative form of Ubc12 (the Nedd8 E2) in U20S cells. This caused an 89% reduction in cell growth compared to wild type [20,48]. Arabidopsis plants with single gene defects in the RUB conjugation pathway are not as severely affected as their mice and fission yeast counterparts [26,29,49]. However, this is probably due to genetic redundancy in this species. There are two closely related isoforms of one subunit of the RUB E1 enzyme called AXR1 and AXL1 and two very similar RUB E2 enzymes called RCE1 and RCE2. Although mutant axr1 plants exhibit a pleiotropic phenotype they are nonetheless viable and fertile. However, the loss of AXR1 and one of the E2 enzymes, RCE1, results in embryonic defects and early lethality [29]. Plants that are deficient in both AXR1 and AXL1 have a similar phenotype (N. Dharmasiri and Estelle, unpublished).
8. Deneddylation The attachment of Nedd8/RUB to cullin is not the only relevant event in Nedd-regulation of cullin function. Recent evidence has shown that the deneddylation of cullin is as important as its attachment. This is most obviously illustrated by the finding that one of the functions of the COP9 signalosome (CSN) is to facilitate removal of Nedd8/RUB from cullin [31,50,51]. 8.1. The COP9 signalosome The CSN was first identified in Arabidopsis in studies of light regulated development [52]. During the intervening years all eight subunits of the CSN have been identified in Arabidopsis as well as S. cerevisiae, S. pombe, C. elegans, Drosophila, and humans [51,53–58]. Strikingly, each of the subunits has significant similarity to subunits of the lid portion of the 26S proteasome. Using yeast two-hybrid and immunoprecipitation experiments, the CSN, and more specifically the CSN2 subunit, has been shown to interact with cullins in humans, fission yeast, and Arabidopsis [31,50,54]. The first evidence that
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the CSN might be involved in deneddylation was the observation that CSN-deficient Arabidopsis plants and S. pombe cells accumulate higher levels of neddylated CUL1 [31,50]. The Deshaies group went on to show that the CSN5 subunit contains a conserved metalloprotease motif called the JAMM motif [59]. The importance of this motif was demonstrated using S. pombe csn5∆ cells deficient in deneddylation activity. The expression of a CSN5 protein with mutations in the JAMM region resulted in the assembly of an intact CSN. However, the mutant protein could not rescue the deneddylation activity in these cells [59]. This result indicates that even though CSN could assemble and presumably bind to cullin, the specific lack of the CSN5/JAMM motif prevented the deneddylation of cullin. Further studies in Arabidopsis and C. elegans indicate that the CSN6 subunit may interact with the RBX1 component of the SCF complex [54,57]. A comprehensive protein–protein interaction analysis of the Arabidopsis CSN subunits concluded that the CSN5/JAMM subunit lies between the CSN2 and CSN6 subunits [54]. When taken together with the known crystal structure of the SCF [13] this allows us to speculate that the interaction of CSN2 and CSN6 with the SCF could serve to position the CSN5 subunit such that it can mediate the cleavage of Nedd8 (Fig. 2). 8.2. DEN1 The mammalian DEN1 thiol cysteine protease was mentioned earlier in this review because of its putative role in Nedd8 maturation [36,60,61]. In addition to this function, DEN1 can also remove Nedd8 from cullin in an in vitro reaction. However, deneddylation is quite inefficient, especially in comparison to the CSN, calling into question the in vivo relevance of this activity. However, unlike the CSN, DEN1 was able to convert hyperneddylated cullin to mono-Nedd8–CUL1. Since DEN1 can efficiently bind both free and hyperneddylated Nedd8–CUL1 but not Nedd8–CUL1, the authors suggest that RBX1 bound to cullin may prevent DEN1 hydrolysis of the covalent bond between Nedd8 and the cullin [13,36]. The authors report that attempts to investigate the effect of depletion of DEN1 on cellular regulation are underway. Concurrent with this study, the human DEN1 orthologue was also identified by Mendoza et al. [61] and termed NEDP (Nedd8 protease). These authors demonstrated that high concentrations of NEDP could cleave Nedd8 from CUL2 in vitro. In addition, expression of NEDP in COS cells resulted in a decrease in modified CUL4A suggesting that NEDP-mediated deneddylation of cullin may be biologically relevant [61]. The precise biological function of DEN1/NEDP awaits further investigation. 8.3. USP21 and NUB1 Chronologically, the first enzyme to be identified with a role in the deneddylation process was the USP21 pepti-
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dase. This enzyme is capable of removing either ubiquitin or Nedd8 from conjugates [32]. Additionally, when USP21 was overexpressed in U20S cells, their growth was decreased by 80% [32]. The same group of researchers also identified a protein they call NUB1 (nedd ultimate buster 1) [20,62]. Overexpression of NUB1 reduces the amount of free and conjugated Nedd8 in COS cells [20]. Sequence analysis revealed that NUB1 contains a PEST domain (found within proteins targeted for degradation) but no isopeptidase motif [20]. This suggests that NUB1 does not enzymatically cleave Nedd8. Rather, the authors suggest that NUB1 and Nedd8 may form a complex that is rapidly degraded. Further studies consistent with this concept showed that inhibition of the proteasome removed the deleterious effect on Nedd8 levels by NUB1 overexpression. Further, NUB1 was shown to interact with the 19S proteasome activator PA700 [63]. The predicted importance of the NUB1–Nedd8 interaction was verified when it was found that in cells overexpressing NUB1, growth was decreased by 83% [20]. Overexpression of either USP21 or NUB1/1L reduce the amount of high molecular weight Nedd8 [20,32,62]. However, it is interesting to note that a putative Nedd conjugate of about 80 kDa remains unaffected [20,32,62]. Since this is the approximate size of Nedd8–CUL1, it is possible that these two proteases may act specifically on Nedd8 that is conjugated to proteins other than cullin. Because no other neddylation target has been identified, it is possible that USP21 and NUB1 may function in the deneddylation of inappropriately formed Nedd8 conjugates. The neddylation pathway is relatively straightforward and well understood. This contrasts with our present knowledge of the deneddylation process in which the precise function of the various players has not been firmly established. Clearly, further study is required to clarify the role of each of these proteins or protein complexes in the regulation of Nedd8–cullin levels.
9. The role of neddylation The process of attachment and removal of Nedd8/RUB is clearly fundamental to the growth of eukaryotic cells. This is exemplified by the numerous examples of aberrant development that result from perturbations in these processes [51]. Additionally, there are many examples in which compromising the neddylation process results in accumulation of important cellular proteins such as the Aux/IAA transcriptional repressors in Arabidopsis, MEI-1/Katanin in C. elegans, and the mammalian CDK inhibitor p27KIP1 [64–66]. Recently, it has become clear that cycles of neddylation and deneddylation are essential for function of the SCF. However, the significance of this cycling remains elusive. Previous experiments suggest that SCF formation can occur independently of Nedd8 attachment and that formation of SCF aids in recruitment of Nedd8 to cullin [67]. How-
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Fig. 2. The role of Nedd8/RUB in SCF function: a speculative model. Nedd8 is activated by its E1 enzyme (A) and passed onto the Nedd8 E2 (Ubc12 in this case—RCE). The charged E2 binds to RBX1, perhaps in competition with CAND1 (B). Nedd8 conjugation to cullin results in CAND1 dissociation permitting formation of the SCF complex (C). A conformational change at the cullin C-terminus allows the ubiquitin E2 to bind the RING finger of RBX1 (D) bringing it into close proximity with its target so that ubiquitin transfer can occur (E). The conformational change caused by Nedd8 binding alters the alignment of bound CSN (F). Following cycles of E2 attachment and poly-ubiquitination the CSN5/JAMM isopeptidase can remove Nedd8 in turn releasing the ubiquitin E2 (G). Either CAND1 or RCE1 is then able to bind SCF depending on the specific cellular requirements (H).
ever, this suggestion is contradicted by more recent models in which dissociation of CAND1 by Nedd8 precludes formation of the SCF [37,38]. One potential solution to this conundrum involves the Nedd8/RUB E2. In humans and Arabidopsis the Nedd8/RUB E2 protein has been shown to interact with RBX1 [29,30]. Therefore, binding of the E2 to RBX1 may itself provoke the dissociation of CAND1 from the cullin subunit and at the same time, promote transfer of Nedd8/RUB to cullin. It will be interesting to determine whether binding of the E2 to RBX1 is able to hinder the association of CAND1 with the C-terminus of cullin. The present evidence allows us to speculate that the role of Nedd8 attachment is to facilitate the recruitment of the ubiquitin E2 to the SCF, thereby allowing efficient ubiquitination to proceed. This hypothesis was first proposed by Kawakami et al. [67] who developed an in vitro system for investigating the effect of Nedd8 attachment to cullin on the ubiquitination of the IB␣ protein [68]. The authors initially showed that ubiquitination of IB␣ could be accelerated by the addition of the neddylation pathway en-
zymes [67]. In this case, neddylation resulted in a significant increase in ubiquitin E2 (Ubc4) binding to the SCF. They also demonstrated that RBX1 had a higher affinity for Ubc4 when in a complex with neddylated CUL1 than with unmodified cullin [67]. Based on these results, the authors proposed that Nedd8 attachment caused a conformational change in cullin that allows the binding of Ubc4 [67]. This hypothesis is supported by the structure of the SCF [13]. In SCFSKP2 , the Nedd8-binding site (Lys720), is positioned just 11 Å from the RBX1 RING domain that binds the ubiquitin E2 [13,69]. Furthermore, mutation of any of six charged residues that create electrostatic surfaces on Nedd8 can diminish the ability of CUL1–RBX1 to facilitate poly-ubiquitination without affecting Nedd8–cullin conjugation [70]. These lines of evidence implicate Nedd8 in the recruitment of the ubiquitin E2 to the CUL–RBX1 subunit of the SCF. Within the last year, a number of models have been proposed concerning the regulation of SCF function by Nedd8, CAND, the CSN and Ubc12/RCE1 [38,51,57]. Fig. 2 illustrates a similar model, incorporating more re-
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cent results, in which cycles of neddylation/deneddylation facilitate the ubiquitination of proteins primed for degradation. Firstly Nedd8/RUB is recruited by a heterodimeric E1 enzyme and subsequently passed onto the Nedd8/RUB E2 (Ubc12 or RCE). This E2 then binds CUL1–RBX1, an interaction that may interfere with binding of CAND1 to cullin. Neddylation proceeds and CAND1 dissociates from CUL–RBX1. Conjugation of Nedd8 may then alter the conformation of the cullin and/or RBX1 such that the RING finger motif of RBX1 is exposed and can interact with the ubiquitin E2. This brings the E2 and its ubiquitin cargo into proximity with its target allowing ubiquitination to occur. Individual CSN subunits have been shown to bind both cullin and RBX1 so it is possible that a conformational change caused by neddylation may also alter the spatial relationship between the CSN and cullin. This could allow the neddylation site to move into close proximity of the JAMM motif within the CSN5 subunit. At this point the CSN removes Nedd8/RUB from the cullin. This could occur immediately after ubiquitin transfer resulting in the E2 being released from the complex, permitting a new round of neddylation [57]. Alternatively, deneddylation may be delayed until the target has been poly-ubiquitinated by cycling of ubiquitin-charged E2 enzymes [51]. The first possibility would appear to be slower since it requires the formation of additional intermediate complexes. Conversely, the second model may allow for more rapid ubiquitination of targets, a scheme that would fit with the extremely brief half-lives of some cellular proteins. In this case the CSN might play a role as a sensor for the SCF and only proceed with its deneddylating activity once a target is sufficiently ubiquitinated. Following deneddylation, the cullin–RBX1 returns to a conformation that is unable to bind the ubiquitin E2 but is susceptible to binding by either Ubc12, if another round of neddylation and ubiquitination is required, or CAND1 if it is not. As with all models this one has limitations. For example, the model holds that Ubc12 interacts with CUL1–RBX1, while the related ubiquitin E2 does not until CUL1 has been neddylated. It will be interesting to see if differences between these two E2 enzymes are responsible for this discrimination. In addition, the model does not take into account the role of the other deneddylating enzymes, such as DEN1 and USP21. Finally, the model is based on studies conducted in a variety of organisms and not all aspects of the model have been demonstrated in a single species. Given the very rapid pace of research in this field, we expect further refinements to the model will occur in the very near future.
Acknowledgements Work in the authors’ lab was supported by grants from the NIH (GM43644), NSF (2010-0115870), and DOE (DE-FG03-99ER20327) to M.E.
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Regulation of cullin-based ubiquitin ligases by the Nedd8/RUB ubiquitin-like proteins Geraint Parry, Mark Estelle∗ Department of Biology, Indiana University, Myers Hall 150, 915 East Third Street, Bloomington, IN 47405, USA
Abstract 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. © 2004 Elsevier Ltd. All rights reserved. Keywords: Nedd8/RUB; Neddylation; Protein degradation; Cullin
1. Introduction In recent years several ubiquitin-like (Ubl) proteins have been identified in eukaryotes. These proteins are covalently attached to target proteins to facilitate a wide variety of cellular processes [1]. The RUB (related to ubiquitin) or Nedd8 (neural precursor cell-expressed developmentally downregulated 8) proteins are conserved throughout eukaryotes and exhibit approximately 55% identity to ubiquitin (Fig. 1a). Arabidopsis RUB1 has 83% identity with murine Nedd8 and 57% with ScRub1p (Fig. 1a) [2]. Nedd8/RUB molecules are post-translational modifiers of cullin proteins, subunits of E3 ubiquitin protein ligases. Despite intensive investigation in diverse species, no other substrate has been identified. A variety of studies have shown that the conjugation of Nedd8/RUB to cullin is important for E3 function. However, the precise function of Nedd8/RUB conjugation remains uncertain. In this article, we will review the pathways of Nedd8/RUB conjugation and deconjugation. Additionally, we will outline the cellular defects associated with loss of these activities and speculate on the role of Nedd8/RUB modification in SCF function. 2. The cullins Members of the cullin family of proteins have been identified in all eukaryotes. A recent phylogenetic analysis ∗ Corresponding
author. Tel.: +1-812-868-1216; fax: +1-812-855-6082. E-mail address: [email protected] (M. Estelle).
1084-9521/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcdb.2003.12.003
of the known eukaryotic cullins established five distinct clades [3]. Each clade contains a cullin protein from humans, Drosophila melanogaster and Caenorhabditis elegans. The Arabidopsis thaliana cullins CUL3A, CUL3B, and CUL4 group loosely with established clades. In contrast, Arabidopsis CUL1, the most highly studied plant cullin, lies together with the closely related Arabidopsis CUL2, outside of the five established cullin clades [3]. Cullins are subunits of at least four related ubiquitin protein ligases; the anaphase promoting complex/cyclosome (APC) [4,5], the SCF complex (SKP1 cullin F-box protein) [6], the VBC (VHL-elongin B-elongin C) [7,8], and a recently identified complex containing CUL3 and a member of the BTB family of proteins (the BTB complex) [9–11]. All cullins are modified by Nedd8/RUB except for the divergent APC subunit Apc2. Although this review will focus on the role of cullin in the SCF complex, it is important to note that Nedd8/RUB has an important role in the VBC and BTB complexes as well. So far the best understood cullin-based E3s are the SCFs. These complexes are comprised of CUL1, an F-box protein (FBP), SKP1, and RBX1 [6]. The function of CUL1 in this complex is to form a scaffold onto which the SKP1–FBP module and RBX1 bind [12,13]. The crucial role of CUL1 and SCFs in cellular regulation has been clearly shown through genetic studies in diverse species. In budding yeast, mutations in CDC53 (the CUL1 gene in this organism) result in cell cycle arrest at the G1/S transition [14]. Similar cell cycle defects as well as changes in mitotic chromosome condensation have been confirmed
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Fig. 1. Sequence alignments of the Nedd8/RUB proteins and the neddylation site on cullin proteins. (a) Nedd8/RUB proteins from Arabidopsis, human, mouse, Drosophila, Caenorhabditis elegans, Saccharomyces pombe, Saccharomyces cerevisiae and the ubiquitin protein from Arabidopsis. (b) Alignment of the C-terminal 200 amino acids of cullin proteins. The conserved Nedd8- and RBX1-binding sites are shown.
following removal of cullin activity in C. elegans [15]. In Arabidopsis, plants that lack CUL1 arrest at a very early stage in embryogenesis [16], as do CUL1-deficient mice [17].
3. Nedd8/RUB A single Nedd8 gene is present in humans, mice, flies, and nematodes. In contrast, Arabidopsis has three RUB genes
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(Fig. 1a). AtRUB1 and AtRUB2 differ at a single amino acid while AtRUB3 is 78% identical to AtRUB1. Unlike AtRUB3 and Nedd8/RUB orthologues in other species, AtRUB1 and AtRUB2 are synthesized as fusion proteins with ubiquitin [18]. Although the significance of this difference is not clear, it is conserved throughout angiosperms and therefore may be important. AtRUB1 and AtRUB2 are expressed in all organs in Arabidopsis while AtRUB3 is expressed only in flower buds and in the stem suggesting that AtRUB3 may have a unique role [2]. Nedd8 is expressed in all mouse tissues examined [19]. Immunohistochemistry of HA-Nedd8 transfected HeLa cells demonstrated that Nedd8 accumulates in the nucleus [20]. This is consistent with the localization of both CUL1 and enzymes in the Nedd8/RUB conjugation pathway to the nucleus in plant and animal species [16,20,21]. Independent crystallization of human Nedd8 and Arabidopsis RUB1 revealed that these proteins contain five -sheets, together with one significant ␣-helix, and two short ␣-helical regions [2,22]. This structure is extremely similar to ubiquitin and SUMO1. As discussed below and elsewhere in this volume, each Ubl is activated by a specific E1 enzyme. Several groups speculate that this specificity could be mediated in part by differences in surface electrostatic potential between Nedd8/RUB and ubiquitin [2,22,23]. In addition, a change from Arg72 in ubiquitin to Ala72 in Nedd8 (or Glu72 in SUMO) may contribute to specificity. This residue is known to be important for interaction with the E1. Further, the change from Arg to Ala corresponds to differences in the side chains of the E1 enzymes at the putative site of interaction. Therefore, Walden et al. [23] hypothesize that specific amino acids in each E1 can interact with the amino acid at residue 72 in their respective Ubl.
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conjugated to a conserved lysine near the C-terminus of the cullin (Fig. 1b). Although the identities of the Nedd8/RUB E1 and E2 have been known for several years, until recently it was not known if an additional E3 activity is required for attachment to the cullin. The main candidate for this role is the RBX1/Roc1 protein, which together with the cullin, is a core component of SCF, VBC, and BTB complexes [9,24–26]. RBX1/Roc1 binds to CUL1 in the C-terminal region of the protein near the site of Nedd8/RUB attachment (Fig. 1b) [13,27]. In vitro studies demonstrated that conjugation of Rub1p to Cdc53p is dependent on the presence of Rbx1p [28]. In more recent experiments, transgenic Arabidopsis that overexpressed RBX1 exhibited a large increase in the amount of modified CUL1, indicating that the RBX1 protein can facilitate attachment of RUB1, potentially as an E3 enzyme [26]. Further, Arabidopsis RBX1 directly binds the RUB E2 enzyme RCE1 suggesting that RBX1 is responsible for bringing the RUB E2, presumably carrying activated RUB, to the cullin protein [29]. Similarly, Morimoto et al. [30] have shown that human Roc1 binds to the Nedd8 E2, Ubc12. This group also demonstrated that a mutation in the RING domain of Roc1 prevents this interaction. Additionally, recombinant Roc1 promotes neddylation of CUL1 in vitro [30]. Unlike the case for most ubiquitin targets, cullin proteins are typically modified by a single Nedd8 molecule. However, several instances of hyperneddylation have been observed [31,32]. During the course of an in vitro neddylation experiment, Wu et al. [12] demonstrated that a small proportion of CUL1 was conjugated to two Nedd8 molecules, It is not clear if this represents a short Nedd8 chain, or Nedd8 attachment to different lysine residues.
5. Nedd8/RUB processing 4. The neddylation pathway Like ubiquitin conjugation, neddylation is accomplished by specific activating (E1) and conjugating (E2) enzymes. These enzymes are similar to those of the ubiquitin pathway except that the Nedd8/RUB E1, like that of other Ubls, consists of two subunits corresponding to N-terminal and C-terminal regions of the ubiquitin E1. The Nedd8/RUB E1 and E2 enzymes have been identified and characterized in many organisms and are listed in Table 1. Nedd8/RUB is
Like ubiquitin, conjugation of Nedd8/RUB requires a carboxyl-glycine [33]. Since all Nedd8/RUB molecules are synthesized with additional amino acids at their C-terminus, these need to be removed prior to attachment to target proteins. A yeast two-hybrid screen with Nedd8 as bait identified a human ubiquitin-C-terminal hydrolase (UHC-L3) [34]. UCH-L3 was shown to interact with both Nedd8 and ubiquitin in vitro but not with other Ubls. Further in vitro analysis proved that UCH-L3, but not the related enzyme
Table 1 Nedd8/RUB activating (E1) and conjugating (E2) enzymes Organism
Nedd8/RUB
E1
E2
Reference
Saccharomyces cerevisiae Saccharomyces pombe Arabidopsis thaliana
ScRub1p SpNedd8 AtRUB1
ENR2, Uba3b SpUba3 AXR1, ECR1
Ubc12 SpUbc12 RCE1
Caenorhabditis elegans Homo sapiens
Ned-8 Nedd8
Ula1, Uba3 APP-BP1, hUba3
Ubc12 hUbc12
Lammer et al. (1997), Liakopoulos et al. (1998) Osaka et al. (2000) Leyser et al. (1993), del Pozo et al. (1998), del Pozo and Estelle (1999), Dharmasiri et al. (2003) Jones and Candido (2000) Osaka et al. (1998), Gong and Yeh (1999)
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UCH-L1, could cleave the C-terminal region of Nedd8 to expose Gly76 [34]. Therefore, UCH-L3 may be involved in the maturation of both ubiquitin and Nedd8. Similarly, the Saccharomyces cerevisiae orthologue of UCH-L3, called Yuh1p, is required for Rub1p C-terminal cleavage and subsequent conjugation to Cdc53p [35]. In contrast to UCH-L3, a recently identified protein from mammalian cells called DEN1 (DENeddylase 1) selectively cleaves Nedd8 [36,60,61]. However, the biological roles of these two enzymes are not known at present.
6. CAND1 A novel protein that acts in the control of SCF function was recently independently discovered by four research groups. This protein is called CAND1 [37], p120CAND1 [38], or TIP120A [39–41]. TIP120A had been described earlier because of its ability to bind the TATA-binding protein [42,43]. In the context of the SCF, CAND1 forms a complex with CUL–RBX1, thus inhibiting the interaction between CUL1 and SKP1. Most revealing, CAND1 preferentially binds deneddylated CUL1 [37,38,40,41]. Further, neddylation of CUL1 promotes the dissociation of CAND1, thus permitting SCF formation [37,38]. Although the four groups agree on most aspects of the CUL1–CAND interaction, Oshikawa et al. [41] report that TIP120A is only able to interact with CUL1 whereas the other groups demonstrate CAND1 binding to other cullins [37–39]. The removal of the C-terminal 31 amino acids from CUL1 prevents CAND1 binding [37,40]. Because this region encompasses the neddylation site, CAND1 may be unable to bind Nedd8–CUL because of steric hindrance due to the presence of Nedd8 near the CAND1-binding site [40]. The converse does not appear to be true as Nedd8 attachment is possible in the presence of CAND1 in HeLa cell extracts [37] and with preformed CUL1–CAND1 complexes in vitro [38]. Additionally, two groups demonstrate that CAND1 binds to an N-terminal region of CUL1, an interaction that prevents SKP1–CUL1 complex formation [37,40]. On the other hand, Oshikawa et al. [41] report that the crucial region for CAND1 binding is the central portion of CUL1. CAND1 affects the neddylation process by binding cullin. In contrast, the recently identified ASPP2 (apoptosis stimulating protein of p53) inhibits the process at its primary step [44]. ASPP2 interacts with APP-BP1, a subunit of the Nedd8 E1. When ASPP2 is expressed in HeLa cells the proportion of unconjugated CUL1 increases suggesting that this protein may have a role in regulation of the Nedd8 pathway.
7. Function of the neddylation pathway The loss of individual cullins results in severe defects in growth and development in all species examined [15–17,45]. In addition, it is clear from experiments in which compo-
nents of the neddylation pathway are removed that cullin function is dependent on its neddylation state. With the exception of the yeast S. cerevisiae, defects in the Nedd8/RUB conjugation pathway result in dramatic phenotypes. In S. pombe, loss of SpNedd8, SpUba3 (E1 subunit), or SpUbc12 (E2) resulted in cell cycle arrest [45]. In C. elegans RNAi mediated reduction in the expression of ceNED-8 and ceUBC-12 results in arrested embryonic development or severe post-embryonic abnormalities [46]. In mice, a defect in cell division is thought to be responsible for the lethality of Uba3 homozygous mice [47]. In utero dissection revealed that the inner cell mass of the mutant embryo underwent premature apoptosis. The mutant trophoblastic cells were unable to enter S phase of the cell cycle [47]. The crucial role of the Nedd8 pathway in humans was demonstrated by overexpression of a dominant negative form of Ubc12 (the Nedd8 E2) in U20S cells. This caused an 89% reduction in cell growth compared to wild type [20,48]. Arabidopsis plants with single gene defects in the RUB conjugation pathway are not as severely affected as their mice and fission yeast counterparts [26,29,49]. However, this is probably due to genetic redundancy in this species. There are two closely related isoforms of one subunit of the RUB E1 enzyme called AXR1 and AXL1 and two very similar RUB E2 enzymes called RCE1 and RCE2. Although mutant axr1 plants exhibit a pleiotropic phenotype they are nonetheless viable and fertile. However, the loss of AXR1 and one of the E2 enzymes, RCE1, results in embryonic defects and early lethality [29]. Plants that are deficient in both AXR1 and AXL1 have a similar phenotype (N. Dharmasiri and Estelle, unpublished).
8. Deneddylation The attachment of Nedd8/RUB to cullin is not the only relevant event in Nedd-regulation of cullin function. Recent evidence has shown that the deneddylation of cullin is as important as its attachment. This is most obviously illustrated by the finding that one of the functions of the COP9 signalosome (CSN) is to facilitate removal of Nedd8/RUB from cullin [31,50,51]. 8.1. The COP9 signalosome The CSN was first identified in Arabidopsis in studies of light regulated development [52]. During the intervening years all eight subunits of the CSN have been identified in Arabidopsis as well as S. cerevisiae, S. pombe, C. elegans, Drosophila, and humans [51,53–58]. Strikingly, each of the subunits has significant similarity to subunits of the lid portion of the 26S proteasome. Using yeast two-hybrid and immunoprecipitation experiments, the CSN, and more specifically the CSN2 subunit, has been shown to interact with cullins in humans, fission yeast, and Arabidopsis [31,50,54]. The first evidence that
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the CSN might be involved in deneddylation was the observation that CSN-deficient Arabidopsis plants and S. pombe cells accumulate higher levels of neddylated CUL1 [31,50]. The Deshaies group went on to show that the CSN5 subunit contains a conserved metalloprotease motif called the JAMM motif [59]. The importance of this motif was demonstrated using S. pombe csn5∆ cells deficient in deneddylation activity. The expression of a CSN5 protein with mutations in the JAMM region resulted in the assembly of an intact CSN. However, the mutant protein could not rescue the deneddylation activity in these cells [59]. This result indicates that even though CSN could assemble and presumably bind to cullin, the specific lack of the CSN5/JAMM motif prevented the deneddylation of cullin. Further studies in Arabidopsis and C. elegans indicate that the CSN6 subunit may interact with the RBX1 component of the SCF complex [54,57]. A comprehensive protein–protein interaction analysis of the Arabidopsis CSN subunits concluded that the CSN5/JAMM subunit lies between the CSN2 and CSN6 subunits [54]. When taken together with the known crystal structure of the SCF [13] this allows us to speculate that the interaction of CSN2 and CSN6 with the SCF could serve to position the CSN5 subunit such that it can mediate the cleavage of Nedd8 (Fig. 2). 8.2. DEN1 The mammalian DEN1 thiol cysteine protease was mentioned earlier in this review because of its putative role in Nedd8 maturation [36,60,61]. In addition to this function, DEN1 can also remove Nedd8 from cullin in an in vitro reaction. However, deneddylation is quite inefficient, especially in comparison to the CSN, calling into question the in vivo relevance of this activity. However, unlike the CSN, DEN1 was able to convert hyperneddylated cullin to mono-Nedd8–CUL1. Since DEN1 can efficiently bind both free and hyperneddylated Nedd8–CUL1 but not Nedd8–CUL1, the authors suggest that RBX1 bound to cullin may prevent DEN1 hydrolysis of the covalent bond between Nedd8 and the cullin [13,36]. The authors report that attempts to investigate the effect of depletion of DEN1 on cellular regulation are underway. Concurrent with this study, the human DEN1 orthologue was also identified by Mendoza et al. [61] and termed NEDP (Nedd8 protease). These authors demonstrated that high concentrations of NEDP could cleave Nedd8 from CUL2 in vitro. In addition, expression of NEDP in COS cells resulted in a decrease in modified CUL4A suggesting that NEDP-mediated deneddylation of cullin may be biologically relevant [61]. The precise biological function of DEN1/NEDP awaits further investigation. 8.3. USP21 and NUB1 Chronologically, the first enzyme to be identified with a role in the deneddylation process was the USP21 pepti-
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dase. This enzyme is capable of removing either ubiquitin or Nedd8 from conjugates [32]. Additionally, when USP21 was overexpressed in U20S cells, their growth was decreased by 80% [32]. The same group of researchers also identified a protein they call NUB1 (nedd ultimate buster 1) [20,62]. Overexpression of NUB1 reduces the amount of free and conjugated Nedd8 in COS cells [20]. Sequence analysis revealed that NUB1 contains a PEST domain (found within proteins targeted for degradation) but no isopeptidase motif [20]. This suggests that NUB1 does not enzymatically cleave Nedd8. Rather, the authors suggest that NUB1 and Nedd8 may form a complex that is rapidly degraded. Further studies consistent with this concept showed that inhibition of the proteasome removed the deleterious effect on Nedd8 levels by NUB1 overexpression. Further, NUB1 was shown to interact with the 19S proteasome activator PA700 [63]. The predicted importance of the NUB1–Nedd8 interaction was verified when it was found that in cells overexpressing NUB1, growth was decreased by 83% [20]. Overexpression of either USP21 or NUB1/1L reduce the amount of high molecular weight Nedd8 [20,32,62]. However, it is interesting to note that a putative Nedd conjugate of about 80 kDa remains unaffected [20,32,62]. Since this is the approximate size of Nedd8–CUL1, it is possible that these two proteases may act specifically on Nedd8 that is conjugated to proteins other than cullin. Because no other neddylation target has been identified, it is possible that USP21 and NUB1 may function in the deneddylation of inappropriately formed Nedd8 conjugates. The neddylation pathway is relatively straightforward and well understood. This contrasts with our present knowledge of the deneddylation process in which the precise function of the various players has not been firmly established. Clearly, further study is required to clarify the role of each of these proteins or protein complexes in the regulation of Nedd8–cullin levels.
9. The role of neddylation The process of attachment and removal of Nedd8/RUB is clearly fundamental to the growth of eukaryotic cells. This is exemplified by the numerous examples of aberrant development that result from perturbations in these processes [51]. Additionally, there are many examples in which compromising the neddylation process results in accumulation of important cellular proteins such as the Aux/IAA transcriptional repressors in Arabidopsis, MEI-1/Katanin in C. elegans, and the mammalian CDK inhibitor p27KIP1 [64–66]. Recently, it has become clear that cycles of neddylation and deneddylation are essential for function of the SCF. However, the significance of this cycling remains elusive. Previous experiments suggest that SCF formation can occur independently of Nedd8 attachment and that formation of SCF aids in recruitment of Nedd8 to cullin [67]. How-
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Fig. 2. The role of Nedd8/RUB in SCF function: a speculative model. Nedd8 is activated by its E1 enzyme (A) and passed onto the Nedd8 E2 (Ubc12 in this case—RCE). The charged E2 binds to RBX1, perhaps in competition with CAND1 (B). Nedd8 conjugation to cullin results in CAND1 dissociation permitting formation of the SCF complex (C). A conformational change at the cullin C-terminus allows the ubiquitin E2 to bind the RING finger of RBX1 (D) bringing it into close proximity with its target so that ubiquitin transfer can occur (E). The conformational change caused by Nedd8 binding alters the alignment of bound CSN (F). Following cycles of E2 attachment and poly-ubiquitination the CSN5/JAMM isopeptidase can remove Nedd8 in turn releasing the ubiquitin E2 (G). Either CAND1 or RCE1 is then able to bind SCF depending on the specific cellular requirements (H).
ever, this suggestion is contradicted by more recent models in which dissociation of CAND1 by Nedd8 precludes formation of the SCF [37,38]. One potential solution to this conundrum involves the Nedd8/RUB E2. In humans and Arabidopsis the Nedd8/RUB E2 protein has been shown to interact with RBX1 [29,30]. Therefore, binding of the E2 to RBX1 may itself provoke the dissociation of CAND1 from the cullin subunit and at the same time, promote transfer of Nedd8/RUB to cullin. It will be interesting to determine whether binding of the E2 to RBX1 is able to hinder the association of CAND1 with the C-terminus of cullin. The present evidence allows us to speculate that the role of Nedd8 attachment is to facilitate the recruitment of the ubiquitin E2 to the SCF, thereby allowing efficient ubiquitination to proceed. This hypothesis was first proposed by Kawakami et al. [67] who developed an in vitro system for investigating the effect of Nedd8 attachment to cullin on the ubiquitination of the IB␣ protein [68]. The authors initially showed that ubiquitination of IB␣ could be accelerated by the addition of the neddylation pathway en-
zymes [67]. In this case, neddylation resulted in a significant increase in ubiquitin E2 (Ubc4) binding to the SCF. They also demonstrated that RBX1 had a higher affinity for Ubc4 when in a complex with neddylated CUL1 than with unmodified cullin [67]. Based on these results, the authors proposed that Nedd8 attachment caused a conformational change in cullin that allows the binding of Ubc4 [67]. This hypothesis is supported by the structure of the SCF [13]. In SCFSKP2 , the Nedd8-binding site (Lys720), is positioned just 11 Å from the RBX1 RING domain that binds the ubiquitin E2 [13,69]. Furthermore, mutation of any of six charged residues that create electrostatic surfaces on Nedd8 can diminish the ability of CUL1–RBX1 to facilitate poly-ubiquitination without affecting Nedd8–cullin conjugation [70]. These lines of evidence implicate Nedd8 in the recruitment of the ubiquitin E2 to the CUL–RBX1 subunit of the SCF. Within the last year, a number of models have been proposed concerning the regulation of SCF function by Nedd8, CAND, the CSN and Ubc12/RCE1 [38,51,57]. Fig. 2 illustrates a similar model, incorporating more re-
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cent results, in which cycles of neddylation/deneddylation facilitate the ubiquitination of proteins primed for degradation. Firstly Nedd8/RUB is recruited by a heterodimeric E1 enzyme and subsequently passed onto the Nedd8/RUB E2 (Ubc12 or RCE). This E2 then binds CUL1–RBX1, an interaction that may interfere with binding of CAND1 to cullin. Neddylation proceeds and CAND1 dissociates from CUL–RBX1. Conjugation of Nedd8 may then alter the conformation of the cullin and/or RBX1 such that the RING finger motif of RBX1 is exposed and can interact with the ubiquitin E2. This brings the E2 and its ubiquitin cargo into proximity with its target allowing ubiquitination to occur. Individual CSN subunits have been shown to bind both cullin and RBX1 so it is possible that a conformational change caused by neddylation may also alter the spatial relationship between the CSN and cullin. This could allow the neddylation site to move into close proximity of the JAMM motif within the CSN5 subunit. At this point the CSN removes Nedd8/RUB from the cullin. This could occur immediately after ubiquitin transfer resulting in the E2 being released from the complex, permitting a new round of neddylation [57]. Alternatively, deneddylation may be delayed until the target has been poly-ubiquitinated by cycling of ubiquitin-charged E2 enzymes [51]. The first possibility would appear to be slower since it requires the formation of additional intermediate complexes. Conversely, the second model may allow for more rapid ubiquitination of targets, a scheme that would fit with the extremely brief half-lives of some cellular proteins. In this case the CSN might play a role as a sensor for the SCF and only proceed with its deneddylating activity once a target is sufficiently ubiquitinated. Following deneddylation, the cullin–RBX1 returns to a conformation that is unable to bind the ubiquitin E2 but is susceptible to binding by either Ubc12, if another round of neddylation and ubiquitination is required, or CAND1 if it is not. As with all models this one has limitations. For example, the model holds that Ubc12 interacts with CUL1–RBX1, while the related ubiquitin E2 does not until CUL1 has been neddylated. It will be interesting to see if differences between these two E2 enzymes are responsible for this discrimination. In addition, the model does not take into account the role of the other deneddylating enzymes, such as DEN1 and USP21. Finally, the model is based on studies conducted in a variety of organisms and not all aspects of the model have been demonstrated in a single species. Given the very rapid pace of research in this field, we expect further refinements to the model will occur in the very near future.
Acknowledgements Work in the authors’ lab was supported by grants from the NIH (GM43644), NSF (2010-0115870), and DOE (DE-FG03-99ER20327) to M.E.
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