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.     

The COP9 signalosome regulates cell proliferation of Dictyostelium discoideum

Regulated protein destruction involving SCF (Skp1/Cullin/F-box, E3 ubiquitin ligase) complexes is required for multicellular development of Dictyostelium discoideum. Dynamic modification of cullin by nedd8 is required for the proper action of SCF. The COP9 signalosome (CSN), first identified in a signaling pathway for light response in plants, functions as a large multi-protein complex that regulates cullin neddylation in eukaryotes. Still, there is extreme sequence divergence of CSN subunits of the yeasts in comparison to the multicellular plants and animals. Using the yeast two-hybrid system, we have identified the CSN5 subunit as a potential interacting partner of a cell surface receptor of Dictyostelium. We further identified and characterized all 8 CSN subunits in Dictyostelium discoideum. Remarkably, despite the ancient origin of Dictyostelium, its CSN proteins cluster very closely with their plant and animal counterparts. We additionally show that the Dictyostelium subunits, like those of other systems are capable of multi-protein interactions within the CSN complex. Our data also indicate that CSN5 (and CSN2) are essential for cell proliferation in Dictyostelium, a phenotype similar to that of multicellular organisms, but distinct from that of the yeasts. Finally, we speculate on a potential role of CSN in cullin function and regulated protein destruction during multicellular development of Dictyostelium.     

Two Distinct Mechanisms Control the Accumulation of Cyclin B1 and Mos in Xenopus Oocytes in Response to Progesterone

Progesterone-induced meiotic maturation of Xenopus oocytes requires the synthesis of new proteins, such as Mos and cyclin B. Synthesis of Mos is thought to be necessary and sufficient for meiotic maturation; however, it has recently been proposed that newly synthesized proteins binding to p34(cdc2) could be involved in a signaling pathway that triggers the activation of maturation-promoting factor. We focused our attention on cyclin B proteins because they are synthesized in response to progesterone, they bind to p34(cdc2), and their microinjection into resting oocytes induces meiotic maturation. We investigated cyclin B accumulation in response to progesterone in the absence of maturation-promoting factor-induced feedback. We report here that the cdk inhibitor p21(cip1), when microinjected into immature Xenopus oocytes, blocks germinal vesicle breakdown induced by progesterone, by maturation-promoting factor transfer, or by injection of okadaic acid. After microinjection of p21(cip1), progesterone fails to induce the activation of MAPK or p34(cdc2), and Mos does not accumulate. In contrast, the level of cyclin B1 increases normally in a manner dependent on down-regulation of cAMP-dependent protein kinase but independent of cap-ribose methylation of mRNA.     

The COP9 signalosome regulates cell proliferation of Dictyostelium discoideum

Regulated protein destruction involving SCF (Skp1/Cullin/F-box, E3 ubiquitin ligase) complexes is required for multicellular development of Dictyostelium discoideum. Dynamic modification of cullin by nedd8 is required for the proper action of SCF. The COP9 signalosome (CSN), first identified in a signaling pathway for light response in plants, functions as a large multi-protein complex that regulates cullin neddylation in eukaryotes. Still, there is extreme sequence divergence of CSN subunits of the yeasts in comparison to the multicellular plants and animals. Using the yeast two-hybrid system, we have identified the CSN5 subunit as a potential interacting partner of a cell surface receptor of Dictyostelium. We further identified and characterized all 8 CSN subunits in Dictyostelium discoideum. Remarkably, despite the ancient origin of Dictyostelium, its CSN proteins cluster very closely with their plant and animal counterparts. We additionally show that the Dictyostelium subunits, like those of other systems are capable of multi-protein interactions within the CSN complex. Our data also indicate that CSN5 (and CSN2) are essential for cell proliferation in Dictyostelium, a phenotype similar to that of multicellular organisms, but distinct from that of the yeasts. Finally, we speculate on a potential role of CSN in cullin function and regulated protein destruction during multicellular development of Dictyostelium.     

Identification of CIP-1-associated regulator of cyclin B (CARB), a novel p21-binding protein acting in the G2 phase of the cell cycle

The cyclin-dependent kinase inhibitor p21(cip1) regulates cell cycle progression, DNA replication, and DNA repair by binding to specific cellular proteins through distinct amino- and carboxyl-terminal protein binding motifs. We have identified a novel human gene, CARB (CIP-1-associated regulator of cyclin B), whose product interacts with the p21 carboxyl terminus. Immunocytochemical analysis demonstrates that the CARB protein is perinuclear and predominantly associated with the centrosome and mitotic spindle poles. In addition, CARB is also able to associate with cyclin B1, a key regulator of mitosis. However, cyclin B1-CARB complex formation occurs preferentially in the absence of p21. Unexpectedly, overexpression of CARB is associated with a growth-inhibitory and ultimately lethal phenotype in p21(-/-) cells but not in p21(+/+) cells. These data identify a novel mechanism that may underlie the effects of p21 in the G(2)/M phases of the cell cycle.     

Direct proteasome inhibition by clasto-lactacystin beta-lactone permits the detection of ubiquitinated p21(waf1) in ML-1 cells.

The ubiquitin proteasome pathway regulates the expression of major cellular regulatory proteins. The ubiquitin proteasome system has been demonstrated to be involved in the expression of the cyclin kinase inhibitor, p21. Ubiquitinated p21 is degraded immediately by 26S proteasome, therefore, the detection of p21 is difficult. We report here an improvement for the detection of ubiquitinated p21 using a proteasome inhibitor, clasto-lactacystin beta-lactone. A p21-enriched cell lysate is obtained by pretreating the cells with deferoxamine to induce p21 mRNA expression followed by treatment with 1x10(-6) M beta-lactone. The concentration of p21 from the cell lysate was performed using an anti-p21 antibody crosslinked to protein G Sepharose. Ubiquitinated p21 was detected on Western blots of the concentrated sample using an anti-ubiquitin antibody. This detection system will be used for further analysis of the regulation of p21 ubiquitination.     

COP9 signalosome function in the DDR

The COP9 signalosome (CSN) is a platform for protein communication in eukaryotic cells. It has an intrinsic metalloprotease that removes the ubiquitin (Ub)-like protein Nedd8 from cullins. CSN-mediated deneddylation regulates culling-RING Ub ligases (CRLs) and controls ubiquitination of proteins involved in DNA damage response (DDR). CSN forms complexes with CRLs containing cullin 4 (CRL4s) which act on chromatin playing crucial roles in DNA repair, checkpoint control and chromatin remodeling. Furthermore, via associated kinases the CSN controls the stability of DDR effectors such as p53 and p27 and thereby the DDR outcome. DDR is a protection against cancer and deregulation of CSN function causes cancer making it an attractive pharmacological target. Here we review current knowledge on CSN function in DDR.     

COP9 signalosome: Discovery,conservation, activity,and function

The COP9 signalosome(CSN)is a conserved protein complex,typically composed of eight subunits(designated as CSN1 to CSN8)in higher eukaryotes such as plants and animals,but of fewer subunits in some lower eukaryotes such as yeasts.The CSN complex is originally identified in plants from a genetic screen for mutants that mimic light-induced photomorphogenic development when grown in the dark.The CSN complex regulates the activity of cullin-RING ligase(CRL)families of E3 ubiquitin ligase complexes,and play critical roles in regulating gene expression,cell proliferation,and cell cycle.This review aims to summarize the discovery,composition,structure,and function of CSN in the regulation of plant development in response to external(light and temperature)and internal cues(phytohormones).     

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 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.     

Annexin 1 regulates cell proliferation by disruption of cell morphology and inhibition of cyclin D1 expression through sustained activation of the ERK1/2 MAPK signal

Cellular proliferation is controlled by the integration and coordination of extracellular signals. This study explores the role of the protein annexin 1 (ANXA1) in the regulation of such events. We show that ANXA1 has a cell-type independent, anti-proliferative function through sustained activation of the ERK signaling cascade. Moreover, ANXA1 reduces proliferation by ERK-mediated disruption of the actin cytoskeleton and ablation of cyclin D1 protein expression and not by ERK-mediated induction of the cyclin-dependent kinase, CDK2, inhibitor p21(cip/waf). Finally, ANXA1 regulates the ERK pathway at a proximal location, by SH2 domain-independent association with the adapter protein Grb-2. In summary, overexpression of ANXA1 mediates the disruption of normal cell morphology and inhibits cyclin D1 expression, therefore reducing cell proliferation through proximal modulation of the ERK signal transduction pathway.     

The COP9 signalosome-mediated deneddylation is stimulated by caspases during apoptosis

In concert with the ubiquitin (Ub) proteasome system (UPS) the COP9 signalosome (CSN) controls the stability of cellular regulators. The CSN interacts with cullin-RING Ub ligases (CRLs) consisting of a specific cullin, a RING protein as Rbx1 and substrate recognition proteins. The Ub-like protein Nedd8 is covalently linked to cullins and removed by the CSN-mediated deneddylation. Cycles of neddylation and deneddylation regulate CRLs. Apoptotic stimuli cause caspase-dependent modifications of the UPS. However, little is known about the CSN during apoptosis. We demonstrate in vitro and in vivo that CSN6 is cleaved most effectively by caspase 3 at D₂₃ after 2–3 h of apoptosis induced by anti-Fas-Ab or etoposide. CSN6 processing occurs in CSN–CRL complexes and is followed by the cleavage of Rbx1, the direct interaction partner of CSN6. Caspase-dependent cutting of Rbx1 is accompanied by decrease of neddylated proteins in Jurkat T cells. Another functional consequence of CSN6 cleavage is the enhancement of CSN-mediated deneddylating activity causing deneddylation of cullin 1 in cells. The CSN-associated deubiquitinating as well as kinase activity remained unchanged in presence of active caspase 3. The cleavage of Rbx1 and increased deneddylation of cullins inactivate CRLs and presumably stabilize pro-apoptotic factors for final apoptotic steps. Bettina K. J. Hetfeld and Andreas Peth contributed equally.     

Hepatic Deficiency of COP9 Signalosome Subunit 8 Induces Ubiquitin-Proteasome System Impairment and Bim-Mediated Apoptosis in Murine Livers

The COP9 signalosome (CSN), an evolutionally highly conserved protein complex composed of 8 unique subunits (CSN1 through CSN8) in higher eukaryotes, is purported to modulate protein degradation mediated by the ubiquitin-proteasome system (UPS) but this has not been demonstrated in a critical mitotic parenchymal organ of vertebrates. Hepatocyte-specific knockout of the Cops8 gene (HS-Csn8KO) was shown to cause massive hepatocyte apoptosis and liver malfunction but the underlying mechanism remains unclear. Here, we report that Csn8/CSN exerts profound impacts on hepatic UPS function and is critical to the stability of the pro-apoptotic protein Bim. Significant decreases in CIS (cytokine-inducible Src homology 2 domain-containing protein), a Bim receptor of a cullin2-based ubiquitin ligase, were found to co-exist with a marked increase of Bim proteins. Csn8 deficiency also significantly decreased 19S proteasome subunit Rpt5 and markedly increased high molecular weight neddylated and ubiquitinated proteins. The use of a surrogate UPS substrate further reveals severe impairment of UPS-mediated proteolysis in HS-Csn8KO livers. Inclusion body-like materials were accumulated in Csn8 deficient hepatocytes. In addition to Bim, massive hepatocyte apoptosis in HS-Csn8KO livers is also associated with elevated expression of other members of the Bcl2 family, including pro-apoptotic Bax as well as anti-apoptotic Bcl2 and Bcl-XL. Increased interaction between Bcl2 and Bim, but not between Bcl2 and Bax, was detected. Hence, it is concluded that hepatic CSN8 deficiency impairs the UPS in the liver and the resultant Bim upregulation likely plays an important role in triggering hepatocyte apoptosis via sequestering Bcl2 away from Bax.     

CSN controls NF-kappaB by deubiquitinylation of IkappaBalpha

The COP9 signalosome (CSN) is a conserved protein complex that regulates assembly and activity of cullin-RING ubiquitin ligases (CRLs). Ubiquitin-dependent degradation of the NF-kappaB inhibitor IkappaBalpha preceeds nuclear translocation of NF-kappaB. For the first time, we show here an inducible interaction of the CSN with IkappaBalpha and that the CSN controls IkappaBalpha and NF-kappaB activity. Strikingly, disruption of the CSN by a small interfering RNA-mediated knockdown of single CSN subunits results in a reduced re-accumulation of IkappaBalpha and prolonged nuclear translocation of NF-kappaB in TNFalpha-stimulated cells. The control of IkappaBalpha by the CSN is regulated by deubiquitinylation of IkappaBalpha conferred by the CSN-associated deubiquitinylase USP15. Protein expression levels of cullin1 and the CRL substrate adapter beta-TrCP are reduced in nonstimulated cells with a disrupted function of the CSN, which might account for an impaired basal turnover of IkappaBalpha. We propose that the CSN controls both CRL activity and stability of the CRL substrate IkappaBalpha. In consequence, basal and signal-induced CRL-dependent turnover of IkappaBalpha is precisely adapted to specific cellular needs.     

Characterization of the role of COP9 signalosome in regulating cullin E3 ubiquitin ligase activity

Cullin RING ligases (CRLs) are the largest family of cellular E3 ubiquitin ligases and mediate polyubiquitination of a number of cellular substrates. CRLs are activated via the covalent modification of the cullin protein with the ubiquitin-like protein Nedd8. This results in a conformational change in the cullin carboxy terminus that facilitates the ubiquitin transfer onto the substrate. COP9 signalosome (CSN)-mediated cullin deneddylation is essential for CRL activity in vivo. However, the mechanism through which CSN promotes CRL activity in vivo is currently unclear. In this paper, we provide evidence that cullin deneddylation is not intrinsically coupled to substrate polyubiquitination as part of the CRL activation cycle. Furthermore, inhibiting substrate-receptor autoubiquitination is unlikely to account for the major mechanism through which CSN regulates CRL activity. CSN also did not affect recruitment of the substrate-receptor SPOP to Cul3, suggesting it may not function to facilitate the exchange of Cul3 substrate receptors. Our results indicate that CSN binds preferentially to CRLs in the neddylation-induced, active conformation. Binding of the CSN complex to active CRLs may recruit CSN-associated proteins important for CRL regulation. The deneddylating activity of CSN would subsequently promote its own dissociation to allow progression through the CRL activation cycle.     

Analysis of cyclin D3-cdk4 complexes in fibroblasts expressing and lacking p27(kip1) and p21(cip1)

Our studies examined the effects of p27(kip1) and p21(cip1) on the assembly and activity of cyclin D3-cdk4 complexes and determined the composition of the cyclin D3 pool in cells containing and lacking these cyclin-dependent kinase inhibitors. We found that catalytically active cyclin D3-cdk4 complexes were present in fibroblasts derived from p27(kip1)-p21(cip1)-null mice and that immunodepletion of extracts of wild-type cells with antibody to p27(kip1) and/or p21(cip1) removed cyclin D3 protein but not cyclin D3-associated activity. Similar results were observed in experiments assaying cyclin D1-cdk4 activity. Data obtained using mixed cell extracts demonstrated that p27(kip1) interacted with cyclin D3-cdk4 complexes in vitro and that this interaction was paralleled by a loss of cyclin D3-cdk4 activity. In p27(kip1)-p21(cip1)-deficient cells, the cyclin D3 pool consisted primarily of cyclin D3 monomers, whereas in wild-type cells, the majority of cyclin D3 molecules were complexed to cdk4 and either p27(kip1) or p21(cip1) or were monomeric. We conclude that neither p27(kip1) nor p21(cip1) is required for the formation of cyclin D3-cdk4 complexes and that cyclin D3-cdk4 complexes containing p27(kip1) or p21(cip1) are inactive. We suggest that only a minor portion of the total cyclin D3 pool accounts for all of the cyclin D3-cdk4 activity in the cell regardless of whether the cell contains p27(kip1) and p21(cip1).