Loss of the CONSTITUTIVE PHOTOMORPHOGENIC9 signalosome subunit 5 is sufficient to cause the cop/det/fus mutant phenotype in Arabidopsis

The COP9 signalosome (CSN) was originally identified based on the constitutively photomorphogenic/de-etiolated/fusca (cop/det/fus) mutants from Arabidopsis thaliana. CSN is evolutionary conserved, and its subunit 5 (CSN5) mediates the deconjugation of NEDD8 from the cullin subunit of E3 ubiquitin ligases (deneddylation). Here, we report on Arabidopsis mutants deficient in CSN5 function. We show that these mutants are phenotypically indistinguishable from the previously described cop/det/fus mutants of other CSN subunits. However, we also show that these mutants retain the CSN complex (lacking CSN5), and this finding is in contrast with the previously described CSN subunit mutants, which lack the CSN complex. We therefore conclude that loss of CSN5 as part of CSN is sufficient to cause the cop/det/fus mutant phenotype. Furthermore, we show that mutants defective in CSN5 as well as mutants defective in CSN are unable to deneddylate the Arabidopsis cullins AtCUL1, AtCUL3A, and AtCUL4. Because these are representative cullin subunits of the three cullin-containing E3 families present in Arabidopsis, we postulate that the cop/det/fus mutant phenotype may be the result of the defects caused by impaired CSN5-dependent deneddylation of cullin-containing E3s.     

TSA1 interacts with CSN1/CSN and may be functionally involved in Arabidopsis seedling development in darkness

The COP9 signalosome (CSN) is a multiprotein complex which participates in diverse cellular and developmental processes.CSN1,one of the subunits of CSN,is essential for assembly of the multiprotein complex via PCI (proteasome,COP9 signalosome and initiation factor 3) domain in the C-terminal half of CSN 1.However,the role of the N-terminal domain (NTD) of CSN 1,which is critical for the function of CSN,is not completely understood.Using a yeast two-hybrid (Y2H) screen,we found that the NTD of CSN1 interacts with TSK-associating protein 1 (TSA1),a reported Ca2+-binding protein.The interaction between CSN1 and TSA1 was confirmed by co-immunoprecipitation in Arabidopsis.tsal mutants exhibited a short hypocotyl phenotype in darkness but were similar to wild-type Arabidopsis under white light,which suggested that TSA1 might regulate Arabidopsis hypocotyl development in the dark.Furthermore,the expression of TSA1 was significantly lower in a csnl null mutant (fus6),while CSN1 expression did not change in a tsal mutant with weak TSA1 expression.Together,these findings suggest a functional relationship between TSA1 and CSN1 in seedling development.     

Structural and Biochemical Characterization of the Cop9 Signalosome CSN5/CSN6 Heterodimer

The Cop9 signalosome complex (CSN) regulates the functional cycle of the major E3 ubiquitin ligase family, the cullin RING E3 ubiquitin ligases (CRLs). Activated CRLs are covalently modified by the ubiquitin-like protein Nedd8 (neural precursor cell expressed developmentally down-regulated protein 8). CSN serves an essential role in myriad cellular processes by reversing this modification through the isopeptidase activity of its CSN5 subunit. CSN5 alone is inactive due to an auto-inhibited conformation of its catalytic domain. Here we report the molecular basis of CSN5 catalytic domain activation and unravel a molecular hierarchy in CSN deneddylation activity. The association of CSN5 and CSN6 MPN (for Mpr1/Pad1 N-terminal) domains activates its isopeptidase activity. The CSN5/CSN6 module, however, is inefficient in CRL deneddylation, indicating a requirement of further elements in this reaction such as other CSN subunits. A hybrid molecular model of CSN5/CSN6 provides a structural framework to explain these functional observations. Docking this model into a published CSN electron density map and using distance constraints obtained from cross-linking coupled to mass-spectrometry, we find that the C-termini of the CSN subunits could form a helical bundle in the centre of the structure. They likely play a key scaffolding role in the spatial organization of CSN and precise positioning of the dimeric MPN catalytic core.     

Neddylation and deneddylation regulate Cul1 and Cul3 protein accumulation

Cullin family proteins organize ubiquitin ligase (E3) complexes to target numerous cellular proteins for proteasomal degradation. Neddylation, the process that conjugates the ubiquitin-like polypeptide Nedd8 to the conserved lysines of cullins, is essential for in vivo cullin-organized E3 activities. Deneddylation, which removes the Nedd8 moiety, requires the isopeptidase activity of the COP9 signalosome (CSN). Here we show that in cells deficient for CSN activity, cullin1 (Cul1) and cullin3 (Cul3) proteins are unstable, and that to preserve their normal cellular levels, CSN isopeptidase activity is required. We further show that neddylated Cul1 and Cul3 are unstable - as suggested by the evidence that Nedd8 promotes the instability of both cullins - and that the unneddylatable forms of cullins are stable. The protein stability of Nedd8 is also subject to CSN regulation and this regulation depends on its cullin-conjugating ability, suggesting that Nedd8-conjugated cullins are degraded en bloc. We propose that while Nedd8 promotes cullin activation through neddylation, neddylation also renders cullins unstable. Thus, CSN deneddylation recycles the unstable, neddylated cullins into stable, unneddylated ones, and promotes cullin-organized E3 activity in vivo.     

The Arabidopsis CSN5A and CSN5B subunits are present in distinct COP9 signalosome complexes, and mutations in their JAMM domains exhibit differential dominant negative effects on development

The COP9 signalosome (CSN) is an evolutionarily conserved multisubunit protein complex involved in a variety of signaling and developmental processes through the regulation of protein ubiquitination and degradation. A known biochemical role attributed to CSN is a metalloprotease activity responsible for the derubylation of cullins, core components for several types of ubiquitin E3 ligases. The CSN's derubylation catalytic center resides in its subunit 5, which in Arabidopsis thaliana is encoded by two homologous genes, CSN5A and CSN5B. Here, we show that CSN5A and CSN5B subunits are assembled into distinct CSN complexes in vivo, which are present in drastically different abundances, with CSN(CSN5A) appearing to be the dominant one. Transgenic CSN5A and CSN5B proteins carrying a collection of single mutations in or surrounding the metalloprotease catalytic center are properly assembled into CSN complexes, but only mutations in CSN5A result in a pleiotropic dominant negative phenotype. The extent of phenotypic effects caused by mutations in CSN5A is reflected at the molecular level by impairment in Cullin1 derubylation. These results reveal that three key metal binding residues as well as two other amino acids outside the catalytic center play important roles in CSN derubylation activity. Taken together, our data provide physiological evidence on a positive role of CSN in the regulation of Arabidopsis SCF (for Skp1-Cullin-F-box) E3 ligases through RUB (for Related to Ubiquitin) deconjugation and highlight the unequal role that CSN(CSN5A) and CSN(CSN5B) play in controlling the cellular derubylation of cullins. The initial characterization of CSN5A and CSN5B insertion mutants further supports these findings and provides genetic evidence on their unequal role in plant development.     

Role of individual subunits of the Neurospora crassa CSN complex in regulation of deneddylation and stability of cullin proteins

The Cop9 signalosome (CSN) is an evolutionarily conserved multifunctional complex that controls ubiquitin-dependent protein degradation in eukaryotes. We found seven CSN subunits in Neurospora crassa in a previous study, but only one subunit, CSN-2, was functionally characterized. In this study, we created knockout mutants for the remaining individual CSN subunits in N. crassa. By phenotypic observation, we found that loss of CSN-1, CSN-2, CSN-4, CSN-5, CSN-6, or CSN-7 resulted in severe defects in growth, conidiation, and circadian rhythm; the defect severity was gene-dependent. Unexpectedly, CSN-3 knockout mutants displayed the same phenotype as wild-type N. crassa. Consistent with these phenotypic observations, deneddylation of cullin proteins in csn-1, csn-2, csn-4, csn-5, csn-6, or csn-7 mutants was dramatically impaired, while deletion of csn-3 did not cause any alteration in the neddylation/deneddylation state of cullins. We further demonstrated that CSN-1, CSN-2, CSN-4, CSN-5, CSN-6, and CSN-7, but not CSN-3, were essential for maintaining the stability of Cul1 in SCF complexes and Cul3 and BTB proteins in Cul3-BTB E3s, while five of the CSN subunits, but not CSN-3 and CSN-5, were also required for maintaining the stability of SKP-1 in SCF complexes. All seven CSN subunits were necessary for maintaining the stability of Cul4-DDB1 complexes. In addition, CSN-3 was also required for maintaining the stability of the CSN-2 subunit and FWD-1 in the SCF(FWD-1) complex. Together, these results not only provide functional insights into the different roles of individual subunits in the CSN complex, but also establish a functional framework for understanding the multiple functions of the CSN complex in biological processes.     

Dual function of Rpn5 in two PCI complexes, the 26S proteasome and COP9 signalosome

Subunit composition and architectural structure of the 26S proteasome lid is strictly conserved between all eukaryotes. This eight-subunit complex bears high similarity to the eukaryotic translation initiation factor 3 and to the COP9 signalosome (CSN), which together define the proteasome CSN/COP9/initiation factor (PCI) troika. In some unicellular eukaryotes, the latter two complexes lack key subunits, encouraging questions about the conservation of their structural design. Here we demonstrate that, in Saccharomyces cerevisiae, Rpn5 plays dual roles by stabilizing proteasome and CSN structures independently. Proteasome and CSN complexes are easily dissected, with Rpn5 the only subunit in common. Together with Rpn5, we identified a total of six bona fide subunits at roughly stoichiometric ratios in isolated, affinity-purified CSN. Moreover, the copy of Rpn5 associated with the CSN is required for enzymatic hydrolysis of Rub1/Nedd8 conjugated to cullins. We propose that multitasking by a single subunit, Rpn5 in this case, allows it to function in different complexes simultaneously. These observations demonstrate that functional substitution of subunits by paralogues is feasible, implying that the canonical composition of the three PCI complexes in S. cerevisiae is more robust than hitherto appreciated.     

The COP9 signalosome interacts with SCF UFO and participates in Arabidopsis flower development

The COP9 signalosome (CSN) is involved in multiple developmental processes. It interacts with SCF ubiquitin ligases and deconjugates Nedd8/Rub1 from cullins (deneddylation). CSN is highly expressed in Arabidopsis floral tissues. To investigate the role of CSN in flower development, we examined the expression pattern of CSN in developing flowers. We report here that two csn1 partially deficient Arabidopsis strains exhibit aberrant development of floral organs, decline of APETALA3 (AP3) expression, and low fertility in addition to defects in shoot and inflorescence meristems. We show that UNUSUAL FLORAL ORGANS (UFO) forms a SCF(UFO) complex, which is associated with CSN in vivo. Genetic interaction analysis indicates that CSN is necessary for the gain-of-function activity of the F-box protein UFO in AP3 activation and in floral organ transformation. Compared with the previously reported csn5 antisense and csn1 null mutants, partial deficiency of CSN1 causes a reduction in the level of CUL1 in the mutant flowers without an obvious defect in CUL1 deneddylation. We conclude that CSN is an essential regulator of Arabidopsis flower development and suggest that CSN regulates Arabidopsis flower development in part by modulating SCF(UFO)-mediated AP3 activation.     

Crystal structure of the human CSN6 MPN domain

The mammalian COP9 signalosome is an eight-subunit (CSN1–CSN8) complex that plays essential roles in multiple cellular and physiological processes. CSN5 and CSN6 are the only two MPN (Mpr1-Pad1-N-terminal) domain-containing subunits in the complex. Unlike the CSN5 MPN domain, CSN6 lacks a metal-binding site and isopeptidase activity. Here, we report the crystal structure of the human CSN6 MPN domain. Each CSN6 monomer contains nine β sheets surrounded by three helices. Two forms of dimers are observed in the crystal structure. Interestingly, a domain swapping of β8 and β9 strands occurs between two neighboring monomers to complete a typical MPN fold. Analyses of the pseudo metal-binding motif in CSN6 suggest that the loss of two key histidine residues may contribute to the lack of catalytic activity in CSN6. Comparing the MPN domain of our CSN6 with that in the CSN complex shows that apart from the different β8–β9 conformation, they have minor conformational differences at two insertion regions (Ins-1 and Ins-2). Besides, the interacting mode of CSN6–CSN6 in our structure is distinct from that of CSN5–CSN6 in the CSN complex structure. Moreover, the functional implications for Ins-1 and Ins-2 are discussed.     

CAND1 enhances deneddylation of CUL1 by COP9 signalosome

Cullin-RING ligases (CRLs) regulate diverse cellular functions such as cell cycle progression and cytokine signaling by ubiquitinating key regulatory proteins. The activity of CRLs is controlled by Nedd8 modification of the cullin subunits. Recent reports have suggested that CAND1, which specifically binds to unmodified CUL1 but not to neddylated one, is required for the in vivo function of SCFs, the CUL1-containing CRLs. We show here that CAND1 and COP9 signalosome (CSN), the major deneddylase of cullins, bind to unneddylated CUL1 in a mutually exclusive way. The suppression of CAND1 expression by small inhibitory RNA enhanced the interaction between CUL1 and CSN, suggesting that CAND1 inhibited the binding of CSN to CUL1. We found that the binding of CSN to CUL1 required the four helix bundle in CUL1 C-terminal domain, which was wrapped around by CAND1 in the CAND1-CUL1-Rbx1 complex. CAND1 greatly facilitated CSN-mediated deneddylation of CUL1 in vitro, which was dependent on its binding to CUL1. Our data suggest that enhancement of CSN-mediated deneddylation by CAND1 may contribute to its function as a positive regulator of SCFs in vivo.     

Deconjugation of Nedd8 from Cul1 Is Directly Regulated by Skp1-F-box and Substrate, and the COP9 Signalosome Inhibits Deneddylated SCF by a Noncatalytic Mechanism

COP9 signalosome (CSN) mediates deconjugation of the ubiquitin-like protein Nedd8 from the cullin subunits of SCF and other cullin-RING ubiquitin ligases (CRLs). This process is essential to maintain the proper activity of CRLs in cells. Here, we report a detailed kinetic characterization of CSN-mediated deconjugation of Nedd8 from SCF. CSN is an efficient enzyme, with a k(cat) of ～1 s(-1) and K(m)for neddylated Cul1-Rbx1 of ～200 nm, yielding a k(cat)/K(m) near the anticipated diffusion-controlled limit. Assembly with an F-box-Skp1 complex markedly inhibited deneddylation, although the magnitude varied considerably, with Fbw7-Skp1 inhibiting by ～5-fold but Skp2-Cks1-Skp1 by only ～15%. Deneddylation of both SCF(Fbw7) and SCF(Skp2-Cks1) was further inhibited ～2.5-fold by the addition of substrate. Combined, the inhibition by Fbw7-Skp1 plus its substrate cyclin E was greater than 10-fold. Unexpectedly, our results also uncover significant product inhibition by deconjugated Cul1, which results from the ability of Cul1 to bind tightly to CSN. Reciprocally, CSN inhibits the ubiquitin ligase activity of deneddylated Cul1. We propose a model in which assembled CRL complexes engaged with substrate are normally refractory to deneddylation. Upon consumption of substrate and subsequent deneddylation, CSN can remain stably bound to the CRL and hold it in low state of reduced activity.     

The COP9 signalosome regulates Skp2 levels and proliferation of human cells

The COP9 signalosome (CSN) is a conserved, multisubunit complex first identified as a developmental regulator in plants. Gene inactivation of single CSN subunits results in early embryonic lethality in mice, indicating that the CSN is essential for mammalian development. The pleiotropic function of the CSN may be related to its ability to remove the ubiquitin-like peptide Nedd8 from cullin-RING ubiquitin ligases, such as the SCF complex, and therefore regulate their activity. However, the mechanism of CSN regulatory action on cullins has been debated, since, paradoxically, the CSN has an inhibitory role in vitro, while genetic evidence supports a positive regulatory role in vivo. We have targeted expression of CSN subunits 4 and 5 in human cells by lentivirus-mediated small hairpin RNA delivery. Down-regulation of either subunit resulted in disruption of the CSN complex and in Cullin1 hyperneddylation. Functional consequences of CSN down-regulation were decreased protein levels of Skp2, the substrate recognition subunit of SCF(Skp2), and stabilization of a Skp2 target, the cyclin-dependent kinase inhibitor p27(Kip1). CSN down-regulation caused an impairment in cell proliferation, which could be partially reversed by suppression of p27(Kip1). Moreover, restoring Skp2 levels in CSN-deficient cells recovered cell cycle progression, indicating that loss of Skp2 in these cells plays an important role in their proliferation defect. Our data indicate that the CSN is necessary to ensure the assembly of a functional SCF(Skp2) complex and therefore contributes to cell cycle regulation of human cells.     

CSN5/Jab1 controls multiple events in the mammalian cell cycle

The COP9 signalosome (CSN) complex is critical for mammalian cell proliferation and survival, but it is not known how the CSN affects the cell cycle. In this study, MEFs lacking CSN5/Jab1 were generated using a CRE-flox system. MEFs ceased to proliferate upon elimination of CSN5/Jab1. Rescue experiments indicated that the JAMM domain of CSN5/Jab1 was essential. CSN5/Jab1-elimination enhanced the neddylation of cullins 1 and 4 and altered the expression of many factors including cyclin E and p53. CSN5/Jab1-elimination inhibited progression of the cell cycle at multiple points, seemed to initiate p53-independent senescence and increased the ploidy of cells. Thus, CSN5/Jab1 controls different events of the cell cycle, preventing senescence and endocycle as well as the proper progression of the somatic cell cycle.

Structured summary

MINT-8046253: Csn1 (uniprotkb:Q99LD4) physically interacts (MI:0914) with Csn5 (uniprotkb:O35864), Csn8 (uniprotkb:Q8VBV7), Csn3 (uniprotkb:O88543), Csn7b (uniprotkb:Q8BV13) and Csn6 (uniprotkb:O88545) by anti bait coimmunoprecipitation (MI:0006)     

The COP9/signalosome increases the efficiency of von Hippel-Lindau protein ubiquitin ligase-mediated hypoxia-inducible factor-alpha ubiquitination

Oxygen-dependent ubiquitination of the alpha-subunit of hypoxia-inducible factor (HIF-alpha) by the (von Hippel-Lindau protein)-Elongin B/C-Cullin2-Rbx1 (VBC-Cul2) ubiquitin ligase, a member of the cullin-RING ubiquitin ligases (CRLs), plays a central role in controlling oxygen metabolism. Nedd8 conjugation of cullins enhances the ligase activity of CRLs, and the COP9/signalosome (CSN) enhances the degradation of several CRL substrates, although it removes Nedd8 from cullins. Here we demonstrate that CSN increased the efficiency of the VBC-Cul2 complex for recognizing and ubiquitinating substrates by facilitating the dissociation of ubiquitinated substrates from the pVHL subunit of the complex. Moreover CSN enhanced HIF-1alpha degradation by promoting the dissociation of HIF-1alpha from pVHL in cells. The length of the polyubiquitin chain conjugated to the substrate appeared to be involved in CSN-mediated dissociation of the substrate from pVHL. In contrast to other mechanisms underlying CSN-mediated activation of CRLs, the dissociation of ubiquitinated substrates from pVHL did not require the deneddylation activity of CSN, implying that CSN enhances degradation of CRL substrates by multiple mechanisms.     

The Minimal Deneddylase Core of the COP9 Signalosome Excludes the Csn6 MPN(-) Domain

The COP9 signalosome (CSN) is a eukaryotic protein complex, which regulates a wide range of biological processes mainly through modulating the cullin ubiquitin E3 ligases in the ubiquitin-proteasome pathway. The CSN possesses a highly conserved deneddylase activity that centers at the JAMM motif of the Csn5 subunit but requires other subunits in a complex assembly. The classic CSN is composed of 8 subunits (Csn1-8), yet in several Ascomycota, the complex is smaller and lacks orthologs for a few CSN subunits, but nevertheless contains a conserved Csn5. This feature makes yeast a powerful model to determine the minimal assemblage required for deneddylation activity. Here we report, that Csi1, a diverged S. cerevisiae CSN subunit, displays significant homology with the carboxyl terminal domain of the canonical Csn6, but lacks the amino terminal MPN(-) domain. Through the comparative and experimental analyses of the budding yeast and the mammalian CSNs, we demonstrate that the MPN(-) domain of the canonical mouse Csn6 is not part of the CSN deneddylase core. We also show that the carboxyl domain of Csn6 has an indispensable role in maintaining the integrity of the CSN complex. The CSN complex assembled with the carboxyl fragment of Csn6, despite its lack of an MPN(-) domain, is fully active in deneddylation of cullins. We propose that the budding yeast Csi1 is a functional equivalent of the canonical Csn6, and thus the composition of the CSN across phyla is more conserved than hitherto appreciated.     

The Organization of a CSN5-containing Subcomplex of the COP9 Signalosome

The COP9 signalosome (CSN) is an evolutionarily conserved multi-protein complex that interfaces with the ubiquitin-proteasome pathway and plays critical developmental roles in both animals and plants. Although some subunits are present only in an ∼320-kDa complex-dependent form, other subunits are also detected in configurations distinct from the 8-subunit holocomplex. To date, the only known biochemical activity intrinsic to the complex, deneddylation of the Cullin subunits from Cullin-RING ubiquitin ligases, is assigned to CSN5. As an essential step to understanding the structure and assembly of a CSN5-containing subcomplex of the CSN, we reconstituted a CSN4-5-6-7 subcomplex. The core of the subcomplex is based on a stable heterotrimeric association of CSN7, CSN4, and CSN6, requiring coexpression in a bacterial reconstitution system. To this heterotrimer, we could then add CSN5 in vitro to reconstitute a quaternary complex. Using biochemical and biophysical methods, we identified pairwise and combinatorial interactions necessary for the formation of the CSN4-5-6-7 subcomplex. The subcomplex is stabilized by three types of interactions: MPN-MPN between CSN5 and CSN6, PCI-PCI between CSN4 and CSN7, and interactions mediated through the CSN6 C terminus with CSN4 and CSN7. CSN8 was also found to interact with the CSN4-6-7 core. These data provide a strong framework for further investigation of the organization and assembly of this pivotal regulatory complex.     

The subunit CSN6 of the COP9 signalosome is cleaved during apoptosis

The COP9 signalosome is a large multiprotein complex that consists of eight subunits termed CSN1-CSN8. The diverse functions of the COP9 complex include regulation of several important intracellular pathways, including the ubiquitin/proteasome system, DNA repair, cell cycle, developmental changes, and some aspects of immune responses. Nod1 is also thought to be an important cytoplasmic receptor involved in innate immune responses. It detects specific motifs of bacterial peptidoglycan, and this results in activation of multiple signaling pathways and changes in cell function. In this report, we performed a yeast two-hybrid screening and discovered that Nod1 interacts with several components of the COP9 signalosome through its CARD domain. Moreover, we observed that activation of the Nod1 apoptotic pathway leads to specific cleavage of the subunit CSN6. This cleavage is concomitant with caspase processing and generates a short amino-terminal peptide of 3 kDa. A complete inhibition of this cleavage was achieved in the presence of the broad spectrum pharmacological inhibitor of apoptosis, Z-VAD. Furthermore, overexpression of CLARP, a specific caspase 8 inhibitor, completely blocked cleavage of CSN6. Taken together, these results suggest a critical role of caspase 8 in the processing of CSN6. Moreover, these findings suggest that CSN6 cleavage may result in modifications of functions of the COP9 complex that are involved in apoptosis.     

The COP9 signalosome (CSN): an evolutionary conserved proteolysis regulator in eukaryotic development

The COP9 signalosome (CSN) is a multiprotein complex of the ubiquitin-proteasome pathway. CSN is typically composed of eight subunits, each of which is related to one of the eight subunits that form the lid of the 26S proteasome regulatory particle. CSN was first identified in Arabidopsis where it is required for the repression of photomorphogenic seedling development in the dark. CSN or CSN-related complexes have by now been reported from most eukaryotic model organisms and CSN has been implicated in a vast array of biological processes. It is widely accepted that CSN directly interacts with cullin-containing E3 ubiquitin ligases, and that CSN is required for their proper function. The requirement of CSN for proper E3 function may at least in part be explained by the observation that CSN subunit 5 (CSN5) is the isopeptidase that deconjugates the essential ubiquitin-like Nedd8 modification from the E3 cullin subunit. In addition to its interaction with E3s, CSN may also regulate proteolysis by its association with protein kinases and deubiquitylating enzymes. This review provides a summary of the role of CSN in regulating protein degradation and in eukaryotic development.     

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

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