The Arabidopsis COP9 signalosome subunit 7 is a model PCI domain protein with subdomains involved in COP9 signalosome assembly

The COP9 Signalosome (CSN) is a multiprotein complex that was originally identified in Arabidopsis thaliana as a negative regulator of photomorphogenesis and subsequently shown to be a general eukaryotic regulator of developmental signaling. The CSN plays various roles, but it has been most often implicated in regulating protein degradation pathways. Six of eight CSN subunits bear a sequence motif called PCI. Here, we report studies of subunit 7 (CSN7) from Arabidopsis, which contains such a motif. Our in vitro and structural results, based on 1.5 A crystallographic data, enable a definition of a PCI domain, built from helical bundle and winged helix subdomains. Using functional binding assays, we demonstrate that the PCI domain (residues 1 to 169) interacts with two other PCI proteins, CSN8 and CSN1. CSN7 interactions with CSN8 use both PCI subdomains. Furthermore, we show that a C-terminal tail outside of this PCI domain is responsible for association with the non-PCI subunit, CSN6. In vivo studies of transgenic plants revealed that the overexpressed CSN7 PCI domain does not assemble into the CSN, nor can it complement a null mutation of CSN7. However, a CSN7 clone that contains the PCI domain plus part of the CSN6 binding domain can complement the null mutation in terms of seedling viability and photomorphogenesis. These transgenic plants, though, are defective in adult growth, suggesting that the CSN7 C-terminal tail plays additional functional roles. Together, the findings have implications for CSN assembly and function, highlighting necessary interactions between subunits.     

Fungal development and the COP9 signalosome

The conserved COP9 signalosome (CSN) multiprotein complex is located at the interface between cellular signaling, protein modification, life span and the development of multicellular organisms. CSN is required for light-controlled responses in filamentous fungi. This includes the circadian rhythm of Neurospora crassa or the repression of sexual development by light in Aspergillus nidulans. In contrast to plants and animals, CSN is not essential for fungal viability. Therefore fungi are suitable models to study CSN composition, activity and cellular functions and its role in light controlled development.     

Roles of COP9 signalosome in cancer

The constitutive photomorphogenesis 9 signalosome (COP9 or CSN) is an evolutionarily conserved multiprotein complex found in plants and animals. Because of the homology between the COP9 signalosome and the 19S lid complex of the proteosome, COP9 has been postulated to play a role in regulating the degradation of polyubiquitinated proteins. Many tumor suppressor and oncogene products are regulated by ubiquitination- and proteosome-mediated protein degradation. Therefore, it is conceivable that COP9 plays a significant role in cancer, regulating processes relevant to carcinogenesis and cancer progression (e.g., cell cycle control, signal transduction and apoptosis). In mammalian cells, it consists of eight subunits (CSN1 to CSN8). The relevance and importance of some subunits of COP9 to cancer are emerging. However, the mechanistic regulation of each subunit in cancer remains unclear. Among the CSN subunits, CSN5 and CSN6 are the only two that each contain an MPN (Mpr1p and Pad1p N-terminal) domain. The deneddylation activity of an MPN domain toward cullin-RING ubiquitin ligases (CRL) may coordinate CRL-mediated ubiquitination activity. More recent evidence shows that CSN5 and CSN6 are implicated in ubiquitin-mediated proteolysis of important mediators in carcinogenesis and cancer progression. Here, we discuss the mechanisms by which some CSN subunits are involved in cancer to provide a much needed perspective regarding COP9 in cancer research, hoping that these insights will lay the groundwork for cancer intervention.     

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.     

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.     

Roles of COP9 signalosome in cancer

The constitutive photomorphogenesis 9 signalosome (COP9 or CSN) is an evolutionarily conserved multiprotein complex found in plants and animals. Because of the homology between the COP9 signalosome and the 19S lid complex of the proteosome, COP9 has been postulated to play a role in regulating the degradation of polyubiquitinated proteins. Many tumor suppressor and oncogene products are regulated by ubiquitination- and proteosome-mediated protein degradation. Therefore, it is conceivable that COP9 plays a significant role in cancer, regulating processes relevant to carcinogenesis and cancer progression (e.g., cell cycle control, signal transduction and apoptosis). In mammalian cells, it consists of eight subunits (CSN1 to CSN8). The relevance and importance of some subunits of COP9 to cancer are emerging. However, the mechanistic regulation of each subunit in cancer remains unclear. Among the CSN subunits, CSN5 and CSN6 are the only two that each contain an MPN (Mpr1p and Pad1p N-terminal) domain. The deneddylation activity of an MPN domain toward cullin-RING ubiquitin ligases (CRL) may coordinate CRL-mediated ubiquitination activity. More recent evidence shows that CSN5 and CSN6 are implicated in ubiquitin-mediated proteolysis of important mediators in carcinogenesis and cancer progression. Here, we discuss the mechanisms by which some CSN subunits are involved in cancer to provide a much needed perspective regarding COP9 in cancer research, hoping that these insights will lay the groundwork for cancer intervention.Key words: ubiquitination, CSN, COP9 signalosome, Mdm2, p53, cancer, MPN domain, neddylation, Nedd8, cullin     

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 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.     

Deletion mutants in COP9/signalosome subunits in fission yeast Schizosaccharomyces pombe display distinct phenotypes

The COP9/signalosome complex is highly conserved in evolution and possesses significant structural similarity to the 19S regulatory lid complex of the proteasome. It also shares limited similarity to the translation initiation factor eIF3. The signalosome interacts with multiple cullins in mammalian cells. In the fission yeast Schizosaccharomyces pombe, the Csn1 subunit is required for the removal of covalently attached Nedd8 from Pcu1, one of three S. pombe cullins. It remains unclear whether this activity is required for all the functions ascribed to the signalosome. We previously identified Csn1 and Csn2 as signalosome subunits in S. pombe. csn1 and csn2 null mutants are DNA damage sensitive and exhibit slow DNA replication. Two further putative subunits, Csn4 and Csn5, were identified from the S. pombe genome database. Herein, we characterize null mutations of csn4 and csn5 and demonstrate that both genes are required for removal of Nedd8 from the S. pombe cullin Pcu1 and that their protein products associate with Csn1 and Csn2. However, neither csn4 nor csn5 null mutants share the csn1 and csn2 mutant phenotypes. Our data suggest that the subunits of the signalosome cannot be considered as a distinct functional unit and imply that different subunits of the signalosome mediate distinct functions.     

Isolation and characterization of high temperature-resistant germination mutants of Arabidopsis thaliana

Temperature is a primary environmental cue for seed germination of many weeds and vegetables. To investigate the mechanism of germination regulation by temperature, we selected five high temperature (thermoinhibition)-resistant germination mutants (TRW lines) from 20,000 T-DNA insertion lines of Arabidopsis. Segregation analyses indicated that each of the five lines had single locus recessive mutations. The seeds of TRW134-15 and TRW187 showed reduced sensitivity to ABA and also to the gibberrellin biosynthesis inhibitor, paclobutrazol. Genetic and nucleotide sequencing analyses indicated that TRW187 is a new allele of abi3 (abi3-14). TRW71-1 exhibited a maternal effect for both thermoinhibition-resistant and transparent testa phenotypes, and genetic analysis revealed that the mutation was allelic to tt7 (tt7-4 sib). Interestingly, the seeds of reduced dormancy mutants rdo1, rdo2, rdo3 and rdo4 were also thermoinhibition tolerant, and all the TRW seeds showed reduced dormancy. Like rdo3, TRW13-1 had shorter siliques and slightly shorter stems than the wild type. The mutation of TRW13-1 was mapped to the bottom arm of chromosome 1 where rdo3 has also been mapped, but the two mutants are not allelic. We designated TRW13-1 as thermoinhibition-resistant germination 1 (trg1). We also mapped the ABA-insensitive mutation of TRW134-15 to the bottom arm of chromosome 5 and named it trg2. These results show that both embryo/endosperm and maternal factors contribute to germination inhibition at supraoptimal temperatures in Arabidopsis. In addition, we confirm the role of ABA in thermoinhibition of seed germination and a link between seed physiological dormancy and response to high temperature.     

The COP9 signalosome and its role in plant development

The COP9 signalosome (CSN) is an evolutionarily conserved multiprotein complex with a role in the regulation of cullin-RING type E3 ubiquitin ligases (CRLs). CSN exerts its function on E3 ligases by deconjugating the ubiquitin-related protein NEDD8 from the CRL cullin subunit. Thereby, CSN has an impact on multiple CRL-dependent processes. In recent years, advances have been made in understanding the structural organisation and biochemical function of CSN: Crystal structure analysis and mass spectrometry-assisted studies have come up with first models of the pair-wise and complex interactions of the 8 CSN subunits. Based on the analysis of mutant phenotypes, it can now be taken as an accepted fact that – at least in plants –the major biochemical function of CSN resides in its deneddylation activity, which is mediated by CSN subunit 5 (CSN5). Furthermore, it could be demonstrated that CSN function and deneddylation are required but not essential for CRL-mediated processes, and models for the role of neddylation and deneddylation in controlling CRL activity are emerging. Significant advances have also been made in identifying pathways that are growth restricting in the Arabidopsis csn mutants. Recently it has been shown that a G2 phase arrest, possibly due to genomic instability, restricts growth in Arabidopsis csn mutants. This review provides an update on recent advances in understanding CSN structure and function and summarises the current knowledge on its role in plant development and cell cycle progression.     

Conservation of the COP9/signalosome in budding yeast

Background

Variation at the PPARG locus may influence susceptibility to type 2 diabetes and related traits. The Pro12Ala polymorphism may modulate receptor activity and is associated with protection from type 2 diabetes. However, there have been inconsistent reports of its association with obesity. The silent C1431T polymorphism has not been as extensively studied, but the rare T allele has also been inconsistently linked to increases in weight. Both rare alleles are in linkage disequilibrium and the independent associations of these two polymorphisms have not been addressed.

Results

We have genotyped a large population with type 2 diabetes (n = 1107), two populations of non-diabetics from Glasgow (n = 186) and Dundee (n = 254) and also a healthy group undergoing physical training (n = 148) and investigated the association of genotype with body mass index. This analysis has demonstrated that the Ala12 and T1431 alleles are present together in approximately 70% of the carriers. By considering the other 30% of individuals with haplotypes that only carry one of these polymorphisms, we have demonstrated that the Ala12 allele is consistently associated with a lower BMI, whilst the T1431 allele is consistently associated with higher BMI.

Conclusion

This study has therefore revealed an opposing interaction of these polymorphisms, which may help to explain previous inconsistencies in the association of PPARG polymorphisms and body weight.     

Consequences of COP9 signalosome and 26S proteasome interaction

The COP9 signalosome (CSN) occurs in all eukaryotic cells. It is a regulatory particle of the ubiquitin (Ub)/26S proteasome system. The eight subunits of the CSN possess sequence homologies with the polypeptides of the 26S proteasome lid complex and just like the lid, the CSN consists of six subunits with PCI (proteasome, COP9 signalosome, initiation factor 3) domains and two components with MPN (Mpr-Pad1-N-terminal) domains. Here we show that the CSN directly interacts with the 26S proteasome and competes with the lid, which has consequences for the peptidase activity of the 26S proteasome in vitro. Flag-CSN2 was permanently expressed in mouse B8 fibroblasts and Flag pull-down experiments revealed the formation of an intact Flag-CSN complex, which is associated with the 26S proteasome. In addition, the Flag pull-downs also precipitated cullins indicating the existence of super-complexes consisting of the CSN, the 26S proteasome and cullin-based Ub ligases. Permanent expression of a chimerical subunit (Flag-CSN2-Rpn6) consisting of the N-terminal 343 amino acids of CSN2 and of the PCI domain of S9/Rpn6, the paralog of CSN2 in the lid complex, did not lead to the assembly of an intact complex showing that the PCI domain of CSN2 is important for complex formation. The consequence of permanent Flag-CSN2 overexpression was de-novo assembly of the CSN complex connected with an accelerated degradation of p53 and stabilization of c-Jun in B8 cells. The possible role of super-complexes composed of the CSN, the 26S proteasome and of Ub ligases in the regulation of protein stability is discussed.     

Mapping salinity tolerance during Arabidopsis thaliana germination and seedling growth

To characterize and dissect genetic variation for salinity tolerance, we assessed variation in salinity tolerance during germination and seedling growth for a worldwide sample of Arabidopsis thaliana accessions. By combining QTL mapping, association mapping and expression data, we identified genomic regions involved in salinity response. Among the worldwide sample, we found germination ability within a moderately saline environment (150 mM NaCl) varied considerable, from >90% among the most tolerant lines to complete inability to germinate among the most susceptible. Our results also demonstrated wide variation in salinity tolerance within A. thaliana RIL populations and identified multiple genomic regions that contribute to this variation. These regions contain known candidate genes, but at least four of the regions contain loci not yet associated with salinity tolerance response phenotypes. Our observations suggest A. thaliana natural variation may be an underutilized resource for investigating salinity stress response.     

The emerging role of the COP9 signalosome in cancer

In the last several years, multiple lines of evidence have suggested that the COP9 signalosome (CSN) plays a significant role in the regulation of multiple cancers and could be an attractive target for therapeutic intervention. First, the CSN plays a key role in the regulation of Cullin-containing ubiquitin E3 ligases that are central mediators of a variety of cellular functions essential during cancer progression. Second, several studies suggest that the individual subunits of the CSN, particularly CSN5, might regulate oncogenic and tumor suppressive functions independently of, or coordinately with, the CSN holocomplex. Thus, deregulation of CSN subunit function can have a dramatic effect on diverse cellular functions, including the maintenance of DNA fidelity, cell cycle control, DNA repair, angiogenesis, and microenvironmental homeostasis that are critical for tumor development. Additionally, clinical studies have suggested that the expression or localization of some CSN subunits correlate to disease progression or clinical outcome in a variety of tumor types. Although the study of CSN function in relation to tumor progression is in its infancy, this review will address current studies in relation to cancer initiation, progression, and potential for therapeutic intervention.     

Making sense of the COP9 signalosome. A regulatory protein complex conserved from Arabidopsis to human

The COP9 signalosome, once defined as a repressor complex of light-activated development in Arabidopsis, has recently been found in humans and is probably present in most multicellular organisms. The COP9 signalosome is closely related to the lid sub-complex of the 26S proteasome in structural composition and probably shares a common evolutionary ancestor. A multifaceted role of the COP9 signalosome in cell-signaling processes is hinted at by its associated novel kinase activity, as well as the involvement of its subunits in regulating multiple cell-signaling pathways and cell-cycle progression. The molecular genetic studies in Arabidopsis suggest that the complex functions as part of a highly conserved regulatory network, whose physiological role in animals remains to be determined.     

Analysis of leaf proteins in late flowering mutants of Arabidopsis thaliana

Late flowering monogenic mutants of Arabidopsis thaliana (L.) Heynh. at the loci co, gi, fca, fve, fwa, fha, fpa, fy and their corresponding wild type, Landsberg erecta , were analysed by two-dimensional gel electrophoresis. All plants were grown under continuous light and proteins were extracted from leaves of the same age (20-day-old). The polypeptide patterns of the mutants at the loci co, gi, fca, fve, fwa, fha, fpa , and Landsberg erecta were identical. The mutant at the fy locus showed a qualitative difference with Landsberg erecta . Crosses were made between this line and the wild type Landsberg erecta . F2 plants, resulting from autopollination of the hybrid, were analysed and showed no cosegregation between the observed protein and the flowering phenotype, indicating that these two lines differ by more than a single mutation.