Fission yeast COP9/signalosome suppresses cullin activity through recruitment of the deubiquitylating enzyme Ubp12p

The COP9/signalosome (CSN) is known to remove the stimulatory NEDD8 modification from cullins. The activity of the fission yeast cullins Pcu1p and Pcu3p is dramatically stimulated when retrieved from csn mutants but inhibited by purified CSN. This inhibition is independent of cullin deneddylation but mediated by the CSN-associated deubiquitylating enzyme Ubp12p, which forms a complex with Pcu3p in a CSN-dependent manner. In ubp12 mutants, as in csn mutants, Pcu3p activity is stimulated. CSN is required for efficient targeting of Ubp12p to the nucleus, where both cullins reside. Finally, the CSN/Ubp12p pathway maintains the stability of the Pcu1p-associated substrate-specific adaptor protein Pop1p. We propose that CSN/Ubp12p-mediated deubiquitylation creates an environment for the safe de novo assembly of cullin complexes by counteracting the autocatalytic destruction of adaptor proteins.     

Ddb1 is required for the proteolysis of the Schizosaccharomyces pombe replication inhibitor Spd1 during S phase and after DNA damage

Recently we showed that the Schizosaccharomyces pombe ddb1 gene plays a role in S phase progression. A mutant S. pombe strain lacking expression of the ddb1 gene exhibited slow replication through both early and late regions causing a slow S phase phenotype. We attributed the phenotypes in the ddb1 strain to an increased activity of the replication checkpoint kinase Cds1. However, the basis for a high basal Cds1 activity in the ddb1 strain was not clear. It was shown that Ddb1 associates with the Cop9/signalosome. Moreover, the phenotypes of the Deltaddb1 strain are remarkably similar to the Deltacsn1 (or Deltacsn2) strain that lacks expression of the Csn1 (or Csn2) subunit of the Cop9/signalosome. Cop9/signalosome cooperates with Pcu4 to induce proteolysis of Spd1, which inhibits DNA replication by inhibiting ribonucleotide reductase. Therefore, we investigated whether Ddb1 is required for the proteolysis of Spd1. Here we show that a S. pombe strain lacking expression of Ddb1 fails to induce proteolysis of Spd1 in S phase and after DNA damage. Moreover, deletion of the spd1 gene attenuates the Cds1 kinase activity in cells lacking the expression of ddb1, suggesting that an accumulation of Spd1 results in the increase of Cds1 activity in the Deltaddb1 strain. In addition, the double mutant lacking spd1 and ddb1 no longer exhibits the growth defects and DNA damage sensitivity observed in the Deltaddb1 strain. Our results establish an essential role of Ddb1 in the proteolysis of Spd1. In addition, the observation provides evidence for a functional link between Ddb1 and the Cop9/signalosome.     

Novel aspects of COP9 signalosome functions revealed through analysis of hypomorphic csn mutants

The COP9 signalosome (CSN) is a conserved eukaryotic protein complex implicated in the regulation of cullin-RING type E3 ubiquitin ligases by cleaving the small peptide RUB/Nedd8 from cullins. However, detailed analysis of CSN physiological functions in Arabidopsis has been hampered by the early seedling-lethality of csn null mutants. We and others have now identified a number of viable hypomorphic csn mutants which start to reveal novel CSN-dependent activities in adult Arabidopsis plants.¹ Here, we present a detailed comparative analysis of the csn5a-1 and csn2-5 mutants as a mean to improve understanding of CSN functions in plant cells. Our observations point to CSN-independent activities of CSN5 and suggest a role of the CSN in cytoskeleton assembly/organization.Key words: Arabidopsis, root skewing, CSN, COP9 signalosome, SCF, ubiquitin, TIR1, auxin     

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.     

The Arabidopsis COP9 signalosome is essential for G2 phase progression and genomic stability

The COP9 signalosome (CSN) is required for the full activity of cullin-RING E3 ubiquitin ligases (CRLs) in eukaryotes. CSN exerts its function on CRLs by removing the ubiquitin-related NEDD8 conjugate from the cullin subunit of CRLs. CSN seems, thereby, to control CRL disassembly or CRL subunit stability. In Arabidopsis thaliana, loss of CSN function leads to constitutive photomorphogenic (cop) seedling development and a post-germination growth arrest. The underlying molecular cause of this growth arrest is currently unknown. Here, we show that Arabidopsis csn mutants are delayed in G2 phase progression. This cell cycle arrest correlates with the induction of the DNA damage response pathway and is suggestive of the activation of a DNA damage checkpoint. In support of this hypothesis, we detected gene conversion events in csn mutants that are indicative of DNA double-strand breaks. DNA damage is also apparent in mutants of the NEDD8 conjugation pathway and in mutants of the E3 ligase subunits CULLIN4, COP1 and DET1, which share phenotypes with csn mutants. In summary, our data suggest that Arabidopsis csn mutants undergo DNA damage, which might be the cause of the delay in G2 cell cycle progression.     

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.     

JAMM: a metalloprotease-like zinc site in the proteasome and signalosome

The JAMM (JAB1/MPN/Mov34 metalloenzyme) motif in Rpn11 and Csn5 underlies isopeptidase activities intrinsic to the proteasome and signalosome, respectively. We show here that the archaebacterial protein AfJAMM possesses the key features of a zinc metalloprotease, yet with a distinct fold. The histidine and aspartic acid of the conserved EX_nHS/THX₇SXXD motif coordinate a zinc, whereas the glutamic acid hydrogen-bonds an aqua ligand. By analogy to the active site of thermolysin, we predict that the glutamic acid serves as an acid-base catalyst and the second serine stabilizes a tetrahedral intermediate. Mutagenesis of Csn5 confirms these residues are required for Nedd8 isopeptidase activity. The active site-like architecture specified by the JAMM motif motivates structure-based approaches to the study of JAMM domain proteins and the development of therapeutic proteasome and signalosome inhibitors.     

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.     

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.     

COP9 signalosome subunits 4 and 5 regulate multiple pleiotropic pathways in Drosophila melanogaster

The COP9 signalosome (CSN) is an essential eight-subunit repressor of light-regulated development in ARABIDOPSIS: This complex has also been identified in animals, though its developmental role remains obscure. CSN subunits have been implicated in various cellular processes, suggesting a possible role for the CSN as an integrator of multiple signaling pathways. In order to elucidate the function of the CSN in animals, a Drosophila model system has previously been established. Gel-filtration analysis with antibodies against CSN subunits 4, 5 and 7 revealed that these proteins act as a complex in Drosophila that is similar in size to the plant and mammalian complexes. Null mutations in either one of two subunits, CSN4 or CSN5, are larval lethal. Successful embryogenesis appears to be a consequence of maternal contribution of the complex. Biochemical analysis indicates that the different subunits are found in both CSN-dependent and CSN-independent forms, and that these forms are differentially affected by the mutations. Phenotypic characterization of these two mutants indicates that they show both shared and unique phenotypes, which suggest specific roles for each subunit. Both mutants have defective oocyte and embryo patterning, and defects in response to DNA damage, while csn5 mutants develop melanotic tumors and csn4 mutants have phenotypes reminiscent of defects in ecdysone signaling.     

CSN facilitates Cullin-RING ubiquitin ligase function by counteracting autocatalytic adapter instability

The COP9 signalosome (CSN) is known to bind cullin-RING ubiquitin ligases (CRLs) and to promote their activity in vivo. The mechanism of this stimulation has remained enigmatic because CSN's intrinsic and associated enzymatic activities paradoxically inhibit CRL activity in vitro. Reconciling this paradox, we show here that Csn5-catalysed cullin (Cul) deneddylation and Ubp12-mediated deubiquitination cooperate in maintaining the stability of labile substrate adapters, thus facilitating CRL function. Various fission-yeast csn and ubp12 deletion mutants have lower levels of the Cul3p adapter Btb3p. This decrease is due to increased autocatalytic, Cul3p-dependent, ubiquitination and the subsequent degradation of Btb3p. The CSN-Ubp12p pathway also maintains the stability of the Cul1p adapter Pop1p, a mechanism required for the efficient destruction of its cognate substrate Rum1p. Emphasizing the physiological importance of this mechanism, we found that the dispensable csn5 and ubp12 genes become essential for viability when adapter recruitment to Cul1p is compromised. Our data suggest that maintenance of adapter stability is a general mechanism of CRL control by the CSN.     

BTB/POZ domain proteins are putative substrate adaptors for cullin 3 ubiquitin ligases

Cullins (CULs) are subunits of a prominent class of RING ubiquitin ligases. Whereas the subunits and substrates of CUL1-associated SCF complexes and CUL2 ubiquitin ligases are well established, they are largely unknown for other cullin family members. We show here that S. pombe CUL3 (Pcu3p) forms a complex with the RING protein Pip1p and all three BTB/POZ domain proteins encoded in the fission yeast genome. The integrity of the BTB/POZ domain, which shows similarity to the cullin binding proteins SKP1 and elongin C, is required for this interaction. Whereas Btb1p and Btb2p are stable proteins, Btb3p is ubiquitylated and degraded in a Pcu3p-dependent manner. Btb3p degradation requires its binding to a conserved N-terminal region of Pcu3p that precisely maps to the equivalent SKP1/F box adaptor binding domain of CUL1. We propose that the BTB/POZ domain defines a recognition motif for the assembly of substrate-specific RING/cullin 3/BTB ubiquitin ligase complexes.     

COP9 signalosome subunit 8 is required for postnatal hepatocyte survival and effective proliferation

Studies using lower organisms and cultured mammalian cells have revealed that the COP9 signalosome (CSN) has important roles in multiple cellular processes. Conditional gene targeting was recently used to study CSN function in murine T-cell development and activation. Using the Cre-loxP system, here we have achieved postnatal hepatocyte-restricted knockout of the csn8 gene (HR-Csn8KO) in mice. The protein abundance of other seven CSN subunits was differentially downregulated by HR-Csn8KO and the deneddylation of all cullins examined was significantly impaired. Moreover, HR-Csn8KO-induced massive hepatocyte apoptosis and evoked extensive reparative responses in the liver, including marked intralobular proliferation of biliary lineage cells and trans-differentiation and proliferation of the oval cells. However, division of pre-existing hepatocytes was significantly diminished in HR-Csn8KO livers. These findings indicate that Csn8 is essential to the ability of mature hepatocytes to proliferate effectively in response to hepatic injury. The histopathological examinations revealed striking hepatocytomegaly in Csn8-deficient livers. The hepatocyte nuclei were dramatically enlarged and pleomorphic with hyperchromasia and prominent nucleoli, consistent with dysplasia or preneoplastic cellular alteration in HR-Csn8KO mice at 6 weeks. Pericellular and perisinusoid fibrosis with distorted architecture was also evident at 6 weeks. It is concluded that CSN8/CSN is essential to postnatal hepatocyte survival and effective proliferation.     

Recruitment of the inhibitor Cand1 to the cullin substrate adaptor site mediates interaction to the neddylation site

Cand1 inhibits cullin RING ubiquitin ligases by binding unneddylated cullins. The Cand1 N-terminus blocks the cullin neddylation site, whereas the C-terminus inhibits cullin adaptor interaction. These Cand1 binding sites can be separated into two functional polypeptides which bind sequentially. C-terminal Cand1 can directly bind to unneddylated cullins in the nucleus without blocking the neddylation site. The smaller N-terminal Cand1 cannot bind to the cullin neddylation region without C-terminal Cand1. The separation of a single cand1 into two independent genes represents the in vivo situation of the fungus Aspergillus nidulans, where C-terminal Cand1 recruits smaller N-terminal Cand1 in the cytoplasm. Either deletion results in an identical developmental and secondary metabolism phenotype in fungi, which resembles csn mutants deficient in the COP9 signalosome (CSN) deneddylase. We propose a two-step Cand1 binding to unneddylated cullins which initiates at the adaptor binding site and subsequently blocks the neddylation site after CSN has left.     

CSN1 N-terminal-dependent activity is required for Arabidopsis development but not for Rub1/Nedd8 deconjugation of cullins: a structure-function study of CSN1 subunit of COP9 signalosome

The COP9 signalosome (CSN) is a multifunctional protein complex essential for arabidopsis development. One of its functions is to promote Rub1/Nedd8 deconjugation from the cullin subunit of the Skp1-cullin-F-box ubiquitin ligase. Little is known about the specific role of its eight subunits in deneddylation or any of the physiological functions of CSN. In the absence of CSN1 (the fus6 mutant), arabidopsis CSN complex cannot assemble, which destabilizes multiple CSN subunits and contributes, together with the loss of CSN1, to the phenotype of fus6. To distinguish CSN1-specific functions, we attempted to rescue the complex formation with deletion or point-mutation forms of CSN1 expressed as transgenes in fus6. We show that the central domain of CSN1 is critical for complex assembly, whereas the C-terminal domain has a supporting role. By expressing the C231 fragment, which contains the structural information but lacks the presumed functional domain located at the N terminus, we have rescued the complex formation and restored the Rub1/Nedd8 deconjugation activity on cullins (fus6/C231). Nonetheless, fus6/C231 exhibits pleiotropic phenotype, including photomorphogenic defects and growth arrest at seedling stage. We conclude that CSN1 N-terminal domain is not required for the Rub1/Nedd8 deconjugation activity of cullins, but contributes to a significant aspect of CSN functions that are essential for plant development.     

The COP9 signalosome-like complex in S. cerevisiae and links to other PCI complexes

The COP9 signalosome (CSN), the lid subcomplex of the proteasome and translational initiation factor 3 (eIF3) share structural similarities and are often referred to as the PCI family of complexes. In multicellular eukaryotes, the CSN is highly conserved as an 8-subunit complex but in Saccharomyces cerevisiae the complex is rather divergent. We further characterize the composition and properties of the CSN in budding yeast and its interactions with these related complexes. Using the generalized profile method we identified CSN candidates, four with PCI domains: Csn9, Csn10, Pci8/Csn11, and Csn12, and one with an MPN domain, Csn5/Rri1. These proteins and an additional interactor, Csi1, were tested for pairwise interactions by yeast two-hybrid and were found to form a cluster surrounding Csn12. Csn5 and Csn12 cofractionate in a complexed form with an apparent molecular weight of roughly 250kDa. However, Csn5 migrates as a monomer in Deltacsn12 supporting the pivotal role of Csn12 in stabilizing the complex. Confocal fluorescence microscopy detects GFP-tagged Csn5 preferentially in the nucleus, whereas in absence of Csn12, Csn10, Pci8/Csn11, or Csi1, Csn5 is delocalized throughout the cell, indicating that multiple subunits are required for nuclear localization of Csn5. Two CSN subunits, Csn9 and Csi1, interact with the proteasome lid subunit Rpn5. Pci8/Csn11 has previously been shown to interact with eIF3. Together, these results point to a network of interactions between these three structurally similar, yet functionally diverse, complexes.     

Disruption of the COP9 signalosome Csn2 subunit in mice causes deficient cell proliferation,accumulation of p53 and cyclin E,and early embryonic death

Csn2 (Trip15/Cops2/Alien) encodes the second subunit of the COP9 signalosome (CSN), an eight-subunit heteromeric complex homologous to the lid subcomplex of the 26S proteasome. CSN is a regulator of SCF (Skp1-cullin-F-box protein)ubiquitin ligases, mostly through the enzymatic activity that deconjugates the ubiquitin-like protein Nedd8 from the SCF Cul1 component. In addition, CSN associates with protein kinase activities targeting p53, c-Jun, and IkappaB for phosphorylation. Csn2 also interacts with and regulates a subset of nuclear hormone receptors and is considered a novel corepressor. We report that targeted disruption of Csn2 in mice caused arrest of embryo development at the peri-implantation stage. Csn2(-/-) blastocysts failed to outgrow in culture and exhibited a cell proliferation defect in inner cell mass, accompanied by a slight decrease in Oct4. In addition, lack of Csn2 disrupted the CSN complex and resulted in a drastic increase in cyclin E, supporting a role for CSN in cooperating with the SCF-ubiquitin-proteasome system to regulate protein turnover. Furthermore, Csn2(-/-) embryos contained elevated levels of p53 and p21, which may contribute to premature cell cycle arrest of the mutant.     

The proto-oncogene Int6 is essential for neddylation of Cul1 and Cul3 in Drosophila

Int6 is a proto-oncogene implicated in various types of cancer, but the mechanisms underlying its activity are not clear. Int6 encodes a subunit of the eukaryotic translation initiation factor 3, and interacts with two related complexes, the proteasome, whose activity is regulated by Int6 in S. pombe, and the COP9 signalosome. The COP9 signalosome regulates the activity of Cullin-Ring Ubiquitin Ligases via deneddylation of their cullin subunit. We report here the generation and analysis of two Drosophila mutants in Int6. The mutants are lethal demonstrating that Int6 is an essential gene. The mutant larvae accumulate high levels of non-neddylated Cul1, suggesting that Int6 is a positive regulator of cullin neddylation. Overexpression in Int6 in cell culture leads to accumulation of neddylated cullins, further supporting a positive role for Int6 in regulating neddylation. Thus Int6 and the COP9 signalosome play opposing roles in regulation of cullin neddylation.