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PubMed2003

The COP9 signalosome: an assembly and maintenance platform for cullin ubiquitin ligases?

The COP9 signalosome (CSN) is a highly conserved protein complex implicated in diverse biological functions that involve ubiquitin-mediated proteolysis. Paradoxically, conserved enzymatic activities associated with CSN inhibit cullin ubiquitin ligase activity in vitro, whereas mutational analysis suggests that CSN promotes cullin-dependent proteolysis in vivo. This apparent paradox can be resolved in a model that proposes CSN-mediated cullin inhibition is a prerequisite for the proper assembly and maintenance of active cullin ubiquitin ligase complexes.

Animals↗

PubMed2000

The COP9 signalosome: from light signaling to general developmental regulation and back.

The COP9 signalosome has eight core subunits that are highly conserved between plants and animals. Some of the subunits in Arabidopsis are found in forms that are independent of the complex. The COP9 complex is essential for animal development. The COP9 signalosome may have both an evolutionary and a physical relationship with both the regulatory lid of the proteasome and eIF3.

COP9 Signalosome Complex↗

PubMed2004

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.

Animals↗

PubMed2001

A role of Arabidopsis COP9 signalosome in multifaceted developmental processes revealed by the characterization of its subunit 3.

The COP9 signalosome is a highly conserved eight-subunit protein complex initially defined as a repressor of photomorphogenic development in Arabidopsis. It has recently been suggested that the COP9 signalosome directly interacts and regulates SCF type E3 ligases, implying a key role in ubiquitin-proteasome mediated protein degradation. We report that Arabidopsis FUS11 gene encodes the subunit 3 of the COP9 signalosome (CSN3). The fus11 mutant is defective in the COP9 signalosome and accumulates significant amount of multi-ubiquitinated proteins. The same mutant is specifically impaired in the 26S proteasome-mediated degradation of HY5 but not PHYA, indicating a selective involvement in protein degradation. Reduction-of-function transgenic lines of CSN3 produced through gene co-suppression also accumulate multi-ubiquitinated proteins and exhibit diverse developmental defects. This result substantiates a hypothesis that the COP9 signalosome is involved in multifaceted developmental processes through regulating proteasome-mediated protein degradation.

Amino Acid Sequence↗

PubMed2006

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.

COP9 Signalosome Complex↗

PubMed2005

Purification method of the COP9 signalosome from human erythrocytes.

The COP9 signalosome (CSN) is a multimeric protein complex that occurs in all eukaryotic cells. Originally described in plants as a regulator of photomorphogenesis, its purification and characterization from mammalian cells revealed significant sequence homologies to subunits of the 26S proteasome lid complex, as well as of the eukaryotic translation initiation factor 3. Recent studies disclosed its participation in processes such as DNA repair, cell cycle regulation, development, and angiogenesis. At the moment, the pleiotropic effects of the CSN point to a regulatory role in the ubiquitin/26S proteasome system, but its exact function still remains to be clarified. This chapter describes the method to purify human CSN from red blood cells. Two outdated erythrocyte concentrates are sufficient to prepare approximately 0.5 mg of CSN. Washed cells are first lysed and then proteins are separated by a DEAE anion-exchange column. The CSN-containing fractions are pooled and subjected to an ammonium sulfate precipitation followed by dialysis. The concentrated proteins are then loaded onto a glycerol density gradient and ultracentrifugation is performed. The purification procedure is continued using two succeeding anion-exchange columns, resulting in a sufficiently pure CSN complex. Optionally, an additional density gradient centrifugation can be attached. The purified CSN complex possesses kinase, deneddylase, and deubiquitinase activities and can be stored for at least 2 months on ice at 4 degrees .

Blotting, Western↗

PubMed2001

Promotion of NEDD-CUL1 conjugate cleavage by COP9 signalosome.

SCF ubiquitin ligases control various processes by marking regulatory proteins for ubiquitin-dependent proteolysis. To illuminate how SCF complexes are regulated, we sought proteins that interact with the human SCF component CUL1. The COP9 signalosome (CSN), a suppressor of plant photomorphogenesis, associated with multiple cullins and promoted cleavage of the ubiquitin-like protein NEDD8 from Schizosaccharomyces pombe CUL1 in vivo and in vitro. Multiple NEDD8-modified proteins uniquely accumulated in CSN-deficient S. pombe cells. We propose that the broad spectrum of activities previously attributed to CSN subunits--including repression of photomorphogenesis, activation of JUN, and activation of p27 nuclear export--underscores the importance of dynamic cycles of NEDD8 attachment and removal in biological regulation.

3T3 Cells↗

PubMed2005

Transactivation of Schizosaccharomyces pombe cdt2+ stimulates a Pcu4-Ddb1-CSN ubiquitin ligase.

Cullin-4 forms a scaffold for multiple ubiquitin ligases. In Schizosaccharomyces pombe, the Cullin-4 homologue (Pcu4) physically associates with Ddb1 and the COP9 signalosome (CSN). One target of this complex is Spd1. Spd1 regulates ribonucleotide reductase (RNR) activity. Spd1 degradation during S phase, or following DNA damage of G2 cells, results in the nuclear export of the small RNR subunit. We demonstrate that Cdt2, an unstable WD40 protein, is a regulatory subunit of Pcu4-Ddb1-CSN ubiquitin ligase. cdt2 deletion stabilises Spd1 and prevents relocalisation of the small RNR subunit from the nucleus to the cytoplasm. cdt2+ is periodically transcribed by the Cdc10/DSC1 transcription factor during S phase and transiently transcribed following DNA damage of G2 cells, corresponding to Spd1 degradation profiles. Cdt2 co-precipitates with Spd1, and Cdt2 overexpression results in constitutive Spd1 degradation. We propose that Cdt2 incorporation into the Pcu4-Ddb1-CSN complex prompts Spd1 targeting and subsequent degradation and that Cdt2 is a WD40 repeat adaptor protein for Cullin-4-based ubiquitin ligase.

Adaptor Proteins, Signal Transducing↗

PubMed2003

COP9 signalosome: a multifunctional regulator of SCF and other cullin-based ubiquitin ligases.

COP9 Signalosome (CSN) is a fascinating protein complex whose biochemical and physiological functions are only beginning to be understood. It is conserved throughout eukaryotes and is critical to the proper development of all multicellular organisms in which its function has been explored. Recent work suggests that CSN plays a key role in sustaining the activity of SCF and other cullin-based ubiquitin ligases, which may account for its essential roles in development. Here, we summarize what is known about CSN, and discuss hypotheses for how CSN promotes the activity of SCF ubiquitin ligases.

Animals↗

PubMed2005

Degradation of the Neurospora circadian clock protein FREQUENCY through the ubiquitin-proteasome pathway.

Phosphorylation of the Neurospora circadian clock protein FREQUENCY (FRQ) promotes its degradation through the ubiquitin-proteasome pathway. Ubiquitination of FRQ requires FWD-1 (F-box/WD-40 repeat-containing protein-1), which is the substrate-recruiting subunit of an SCF (SKP/Cullin/F-box)-type ubiquitin ligase. In the fwd-1 mutant strains, FRQ degradation is defective, resulting in the accumulation of hyperphosphorylated FRQ and the loss of the circadian rhythmicities. The CSN (COP9 signalosome) promotes the function of SCF complexes in vivo. But in vitro, deneddylation of cullins by CSN inhibits SCF activity. In Neurospora, the disruption of the csn-2 subunit impairs FRQ degradation and compromises the normal circadian functions. These defects are due to the dramatically reduced levels of FWD-1 in the csn-2 mutant, a result of its rapid degradation. Other components of the SCF(FWD-1) complex, SKP-1 and CUL-1 are also unstable in the mutant. These results establish important roles for SCF(FWD-1) and CSN in the circadian clock of Neurospora and suggest that they are conserved components of the eukaryotic circadian clocks. In addition, these findings resolve the CSN paradox and suggest that the major function of CSN is to maintain the stability of SCF ubiquitin ligases in vivo.

Biological Clocks↗

PubMed2002

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.

Amino Acid Sequence↗

PubMed2002

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.

Animals↗

PubMed2004

Expression pattern of the JAB1/CSN5 gene during murine embryogenesis: colocalization with NEDD8.

The COP9 signalosome (CSN) is a conserved multiprotein complex, with an important developmental role in several organisms, ranging from plants to mammalians. The influence of the CSN on several signaling and developmental processes has been ascribed to its ability to regulate degradation of a number of signaling proteins by the ubiquitin-proteasome system. The CSN controls the function of the SCF ubiquitin-ligase complex through an enzymatic activity that removes the small ubiquitin-like molecule NEDD8 from the cullin component of the SCF and that requires subunit 5 of the CSN (JAB1/CSN5). Mutants of the CSN display early embryonic lethality, a feature that has hindered further characterization of the role of the CSN at later stages of mammalian development. Here we report the analysis of JAB1/CSN5 expression pattern in the mouse embryo. At early stages of development, JAB1/CSN5 transcripts were present with low expression levels in all tissues. Preferential expression in selected tissues was detected starting at E11.5, with higher levels in dorsal root ganglia; at later stages, prominent expression of JAB1/CSN5 transcripts was observed in cranial nerve, spinal and sympathetic ganglia, as well as in selected epithelia, such as the oral and the olfactory epithelium. In the adult brain, additional areas of JAB1/CSN5 expression were the hippocampus and the Purkinjie layer of the cerebellum. We also analyzed the temporal and spatial expression pattern of NEDD8, and found that it substantially overlapped JAB1/CSN5 expression at all stages analyzed, supporting the model of a functional interaction between the two proteins during developmental processes.

Animals↗

PubMed2005

The zinc finger of the CSN-associated deubiquitinating enzyme USP15 is essential to rescue the E3 ligase Rbx1.

The COP9 signalosome (CSN) is a conserved protein complex found in all eukaryotic cells and involved in the regulation of the ubiquitin (Ub)/26S proteasome system. It binds numerous proteins, including the Ub E3 ligases and the deubiquitinating enzyme Ubp12p, the S. pombe ortholog of human USP15. We found that USP15 copurified with the human CSN complex. Isolated CSN complex exhibited protease activity that deubiquitinated poly-Ub substrates and was completely inhibited by o-phenanthroline (OPT), a metal-chelating agent. Surprisingly, the recombinant USP15 was also not able to cleave isopeptide bonds of poly-Ub chains in presence of OPT. Detailed analysis of USP sequences led to the discovery of a novel zinc (Zn) finger in USP15 and related USPs. Mutation of a single conserved cysteine residue in the predicted Zn binding motif resulted in the loss of USP15 capability to degrade poly-Ub substrates, indicating that the Zn finger is essential for the cleavage of poly-Ub chains. Moreover, pulldown experiments demonstrated diminished binding of tetra-Ub to mutated USP15. Cotransfection of USP15 and the Ub ligase Rbx1 revealed that the wild-type deubiquitinating enzyme, but not the USP15 mutant with a defective Zn finger, stabilized Rbx1 toward the Ub system, most likely by reversing poly/autoubiquitination. In summary, a functional Zn finger of USP15 is needed to maintain a conformation essential for disassembling poly-Ub chains, a prerequisite for rescuing the E3 ligase Rbx1.

Amino Acid Sequence↗

PubMed2005

Purification of the COP9 signalosome from porcine spleen, human cell lines, and Arabidopsis thaliana plants.

COP9 signalosome (CSN) is an evolutionarily conserved multisubunit protein complex involved in diverse cellular and developmental processes in eukaryotes. CSN functions in the cell as proteases that deconjugate Nedd8/Rub1 from cullin family proteins and depolymerize ubiquitin chains. As such, CSN represents an important regulator of multiple cullin-based E3 ubiquitin ligases. CSN has also been shown to associate with protein kinase activities. This chapter describes purification of the CSN complex by classical chromatography from porcine spleen and by immunoaffinity purification procedures from cultured human cells and transgenic Arabidopsis plants expressing epitope-tagged CSN subunits. It also describes in vitro deneddylation assays using the HeLa cell extract or the Arabidopsis cell extract, which we have used to test and compare the activity of purified CSN complexes.

Animals↗

PubMed2001

Arabidopsis eIF3e (INT-6) associates with both eIF3c and the COP9 signalosome subunit CSN7.

The Arabidopsis COP9 signalosome is a multisubunit repressor of photomorphogenesis that is conserved among eukaryotes. This complex may have a general role in development. As a step in dissecting the biochemical mode of action of the COP9 signalosome, we determined the sequence of proteins that copurify with this complex. Here we describe the association between components of the COP9 signalosome (CSN1, CSN7, and CSN8) and two subunits of eukaryotic translation initiation factor 3 (eIF3), eIF3e (p48, known also as INT-6) and eIF3c (p105). To obtain a biochemical marker for Arabidopsis eIF3, we cloned the Arabidopsis ortholog of the eIF3 subunit eIF3b (PRT1). eIF3e coimmunoprecipitated with CSN7, and eIF3c coimmunoprecipitated with eIF3e, eIF3b, CSN8, and CSN1. eIF3e directly interacted with CSN7 and eIF3c. However, eIF3e and eIF3b cofractionated by gel filtration chromatography in a complex that was larger than the COP9 signalosome. Whereas eIF3, as detected through eIF3b, localized solely to the cytoplasm, eIF3e, like CSN7, was also found in the nucleus. This suggests that eIF3e and eIF3c are probably components of multiple complexes and that eIF3e and eIF3c associate with subunits of the COP9 signalosome, even though they are not components of the COP9 signalosome core complex. This interaction may allow for translational control by the COP9 signalosome.

Animals↗

PubMedDate not supplied

COPing with hypoxia.

To understand how cells respond to altered oxygenation, a frequent experimental paradigm is to isolate known components of bona fide oxygen responsive proteins. Recent studies have shown that a protein known as CSN5 or JAB1 interacts with both the HIF-1alpha oxygen-responsive transcription factor and its oxygen-dependent regulator, the Von Hippel-Lindau (pVHL) tumor suppressor. CSN5 is a component of the COP9 Signalosome (CSN) which is a multi-subunit protein that has high homology to the lid of the 19S lid of 26S proteasome. The exact function of the CSN5 interaction with pVHL and HIF-1alpha remains to be fully elucidated, but it is clear that the interaction is both oxygen dependent and that CSN5 may play different roles under oxic and hypoxic responses. Further, evidence has also been published indicating that pVHL can be potentially post-translationally modified by CSN5 (de-neddylation) and that CSN5 transcription is regulated by hypoxia as are many of the key pVHL/HIF-1alpha regulatory genes such as the PHDs and OS-9. This review will give a broad overview of known CSN5 and COP9 Signalosome functions and how these functions impact the pVHL/HIF-1alpha signaling complex and potentially other oxygen-sensitive response networks.

Amino Acid Sequence↗

PubMed2003

On again-off again: COP9 signalosome turns the key on protein degradation.

The COP9 signalosome is an eight-subunit protein complex that regulates protein ubiquitination and protein turnover in a variety of plant developmental and physiological contexts, including light-regulated development, hormone signaling, and defense against pathogens. In all eukaryotes tested, the COP9 signalosome is able to posttranslationally modify the cullin subunit of E3-ubiquitin-ligase complexes by cleaving off the covalently coupled peptide, Nedd8. Two contrasting models ascribe stimulatory or inhibitory roles to the modification of cullin/E3 that is mediated by the COP9 signalosome. There is considerable disagreement as to whether Nedd8 cleavage underlies all of the COP9 signalosome's numerous cellular and phenotypic effects. This is because macroscopic phenotypes do not always correlate with biochemical defects in COP9 signalosome mutants. Additional biochemical activities, including protein interactions with the cellular machineries for protein phosphorylation, protein turnover, and protein translation, have been proposed to account for the role of the COP9 signalosome in development and disease.

Arabidopsis Proteins↗