Increasing evidence indicates that removal of Nedd8 from its cullin targets via CSN-mediated proteolysis plays a significant role in the regulation of cullin-dependant E3 ubiquitylation. This hypothesis is supported by the observation that deletion of a CSN subunit in S. pombe
resulted in the accumulation of a predominantly neddylated cullin, thus suggesting that the CSN plays a critical role in Nedd8 deconjugation in vivo. Furthermore, addition of purified CSN to neddylated cullins from yeast extracts resulted in deneddylation of the target cullin. Also, in various genetic models (Drosophila
, C. elegans
), CSN activity is required for the deneddylation of various cullins including CUL1, CUL2 and CUL3 [21
]. These results paradoxically suggest that contrary to its inhibition of in vitro ubiquitylation, the CSN is required for the degradation of cellular cullin E3 substrates.
Wolf et al. have postulated that the CSN paradox may reflect that the in vivo function of CSN-mediated cullin inhibition is to ‘license’ CSN for its subsequent activation [53
]. In this model, the licensing mechanism would be similar to the assembly of pre-replication complexes (pre-RCs) at replication origins. In much the same way that low Cdk activity permits pre-RC assembly, they suggest that CSN-mediated cullin inhibition may create an environment conducive to the assembly of new cullin–ubiquitin ligase complexes that are subsequently activated after release from the CSN. Cullin complexes may then have to return to the CSN for re-assembly ().
Cope and Deshaies have also postulated a role for CSN in regulating cycles of SCF–ROC1 assembly [54
]. In this scenario, an active deneddylated cullin-based E3 ligase–Rbx1 complex that is bound to its degradation target recruits the CSN, which cleaves Nedd8 from the cullin. This allows the binding of p120CAND1 (discussed below) to the cullin, which results in the separation of the Ub E3 ligase recognition complex from the cullin. Subsequent neddylation of the cullin by the Rbx1-dependant Ubc12 E2 conjugating enzyme facilitates the dissociation of p120CAND1, resulting in formation of an active Ub E3. This model suggests that cullin neddylation controls cycles of Ub E3 ligase assembly. Additionally, cullin mutants that are neddylation deficient should exhibit constitutive association with p120CAND1, thus exhibiting a significant deficiency in Ub E3 core–cullin–Rbx1 assembly. The Nedd8 E2 conjugating enzyme Ubc12 has also been shown to bind CUL3 independently of the CSN [26
]. This suggests that cullin neddylation occurs following the release of the CSN holocomplex. The regulatory mechanisms facilitating association/dissociation of the CSN from the cullin remain undefined. Also, several critical question such as how CSN-mediated functional inhibition or assembly is regulated and how Ub E3 recognition complexes (i.e. pVHL/elongin C and B) are assembled with the cullin–Rbx1 complex remain unanswered. Thus, given the current models, the CSN would act as a platform that ensures the transient inactivation of cullin complexes by promoting the release of their associated E2s. Therefore, the cycle of neddylation stimulating the association of cullins with Ub E2s would be followed by CSN-mediated deneddylation and Ub E2 dissociation. This would suggest that CSN-mediated cullin deneddylation prevents inappropriate Ub E3 activity. The question of why the CSN can stably associate with both unneddylated and neddylated cullins however, remains a great mystery.
CSN-dependent isopeptidase activity is sensitive to metal ion chelators due to CSN5 containing a conserved, putative metal-binding motif (EXn
SXXD), referred to as the JAMM motif embedded within the larger MPN domain. The JAMM metalloprotease domain within the CSN5 subunit of the CSN is necessary for deneddylase activity, as mutations disrupting this motif accumulate neddylated cullins in S. pombe
]. Furthermore, the integrity of the putative JAMM catalytic domain is required for proper photoreceptor cell development in Drosophila
. These results suggest that the CSN is a metalloprotease whose proteolytic activity is physiologically important. As all fission and budding yeast csn
mutants are deficient in cullin deneddylation, an intact CSN complex seems to be required for deneddylation activity [20
]. Reciprocally, the CSN5 subunit alone is not able to catalyze the deneddylation reaction, suggesting that the deneddylation function requires CSN5 association with other subunits of the CSN complex. Theoretically, CSN isopeptidase activity would also be regulated by the reduced/oxidized state of the CSN5-metalloprotease as hypoxia can directly and indirectly affect the function of other metalloproteases [57
The CSN has also been associated with cullin-based Ub E3 subcellular localization and stability. For instance, CSN5 mutants in Drosophila
accumulate high levels of neddylated CUL1 in the cytoplasm thus suggesting a role for the CSN in influencing the subcellular localization of cullins [25
]. Another example is the observation that in the dark, the putative E3 ligase of the plant transcription factor HY5 (COP1) translocates into the nucleus from the cytoplasm, resulting in degradation of HY5 [17
]. Importantly, nuclear translocation of COP1 requires the CSN, although exactly how CSN targets COP1 to the nucleus remains undefined. Surprisingly, Gemmill et al. also reported that wt pVHL influenced a notable perinuclear accumulation of CSN5 that was not evident in the original mutant pVHL cell line [59
]. It is currently unknown whether the CSN5 observed in this study was a component of the CSN holocomplex or a smaller CSN5-containing complex. LFA-1 engagement also results in nuclear translocation of CSN5 and the subsequent nuclear export of p27 is CSN5-dependent [60
]. It has also been shown that CSN1 affects the subcellular distribution of Suc22 (RNR small subunit) [73
]. These observations suggest that the CSN can mediate subcellular localization of Ub E3 targets and that the Ub E3 targets could reciprocally influence subcellular localization of CSN subunits.
Importantly, a recent study suggested that neddylation is a regulated cellular process. It was shown that the CSN-free DDB2–DDB1–CUL4A–ROC1/Rbx1 complex became associated with chromatin in response to UV [24
]. Concomitantly, a large portion of CUL4A was found to be neddylated. Subsequently, however, the CSN joined the CUL4A ligase–chromatin complex, resulting in the conversion of neddylated CUL4A into its unmodified form. It has also been shown that light and oxygen can also influence CSN assembly with Ub E3 ligases [2
]. These findings imply that CSN-mediated deneddylase activity and/or association with Ub E3 ligases can be regulated by cellular environmental queues.
Several questions stand out in evaluating the models considered here. For instance, what triggers the release and activation of cullin complexes held in custody by the CSN? How do external signals such as sunlight, hypoxia, auxin, and UV regulate the CSN? What is the role of the CSN in nuclear targeting of various proteins? What is the role of CSN sub-complexes in cullin regulation? Finally, what other CSN-associated enzymatic activities are uncharacterized? For example, some CSN subunits associate with 19S proteasome and eIF3 components and there is almost no information concerning the functions associated with these interactions.