JAB1/CSN5 and the COP9 signalosome. A complex situation

The Jun activating binding protein (JAB1) specifically stabilizes complexes of c-Jun or JunD with AP-1 sites, increasing the specificity of target gene activation by AP-1 proteins. JAB1 is also known as COP9 signalosome subunit 5 (CSN5), which is a component of the COP9 signalosome regulatory complex (CSN). Over the past year, JAB1/CSN5 has been implicated in numerous signaling pathways including those that regulate light signaling in plants, larval development in Drosophila, and integrin signaling, cell cycle control, and steroid hormone signaling in a number of systems. However, the general role of the CSN complex, and the specific role of JAB1/CSN5, still remain obscure. This review attempts to integrate the available data to help explain the role of JAB1/CSN5 and the COP9 signalosome in regulating multiple pathways (in this review, both JAB1 and CSN5 terminologies are used interchangeably, depending on the source material).     

Inositol 1,3,4-trisphosphate 5/6-kinase associates with the COP9 signalosome by binding to CSN1

The COP9 signalosome (CSN) is a complex of eight proteins first identified as a repressor of plant photomorphogenesis. A protein kinase activity associated with the COP9 signalosome has been reported but not identified; we present evidence for inositol 1,3,4-trisphosphate 5/6-kinase (5/6-kinase) as a protein kinase associated with the COP9 signalosome. We have shown that 5/6-kinase exists in a complex with the eight-component COP9 signalosome both when purified from bovine brain and when transfected into HEK 293 cells. 5/6-kinase phosphorylates the same substrates as those of the COP9 signalosome, including IkappaBalpha, p53, and c-Jun but fails to phosphorylate several other substrates, including c-Jun 1-79, which are not substrates for the COP9-associated kinase. Both the COP9 signalosome- associated kinase and 5/6-kinase are inhibited by curcumin. The association of 5/6-kinase with the COP9 signalosome is through an interaction with CSN1, which immunoprecipitates with 5/6-kinase. In addition, the inositol kinase activity of 5/6-kinase is inhibited when in a complex with CSN1. We propose that 5/6-kinase is the previously described COP9 signalosome-associated kinase.     

Molecular Characterization of Subunit 6 of the COP9 Signalosome and Its Role in Multifaceted Developmental Processes in Arabidopsis

The COP9 signalosome is a highly conserved protein complex initially identified as a repressor of photomorphogenesis. Here, we report that subunit 6 of the Arabidopsis COP9 signalosome is encoded by a family of two genes (CSN6A and CSN6B) located on chromosomes V and IV, respectively. The CSN6A and CSN6B proteins share 87% amino acid identity and contain a MPR1p and PAD1p N-terminal (MPN) domain at the N-terminal region. The CSN6 proteins share homology with CSN5 and belong to the Mov34 superfamily of proteins. CSN6 proteins present only in the complex form and coimmunoprecipitate with other known subunits of the COP9 signalosome. Partial loss-of-function strains of the COP9 signalosome created by antisense and cosuppression with CSN6A exhibit diverse developmental defects, including homeotic organ transformation, symmetric body organization, and organ boundary definition. Protein blot analysis revealed that the defective plants accumulate significant amounts of ubiquitinated proteins, supporting the conclusion that the COP9 signalosome regulates multifaceted developmental processes through its involvement in ubiquitin/proteasome-mediated protein degradation.     

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.     

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.     

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 subunit 1 of the COP9 signalosome suppresses gene expression through its N-terminal domain and incorporates into the complex through the PCI domain

The COP9 signalosome is a conserved protein complex composed of eight subunits. Individual subunits of the complex have been linked to various signal transduction pathways leading to gene expression and cell cycle control. However, it is not understood how each subunit executes these activities as part of a large protein complex. In this study, we dissected structure and function of the subunit 1 (CSN1 or GPS1) of the COP9 signalosome relative to the complex. We demonstrated that the C-terminal half of CSN1 encompassing the PCI domain is responsible for interaction with CSN2, CSN3, and CSN4 subunits and is required for incorporation of the subunit into the complex. The N-terminal fragment of CSN1 cannot stably associate with the complex but can translocate to the nucleus on its own. We further show that CSN1 or the N-terminal fragment of CSN1 (CSN1-N) can inhibit c-fos expression from either a transfected template or a chromosomal transgene ( fos-lacZ). Moreover, CSN1 as well as CSN1-N can potently suppress signal activation of a AP-1 promoter and moderately suppress serum activation of a SRE promoter, but is unable to inhibit PKA-induced CRE promoter activity. We conclude that the N-terminal half of CSN1 harbors the activity domain that confers most of the repression functions of CSN1 while the C-terminal half allows integration of the protein into the COP9 signalosome.     

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.     

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.     

Dual Regulation of Dendritic Morphogenesis in Drosophila by the COP9 Signalosome

Altered dendritic arborization contributes to numerous physiological processes including synaptic plasticity, behavior, learning and memory, and is one of the most consistent neuropathologic conditions found in a number of mental retardation disorders, schizophrenia, and neurodegenerative disease. COP9 signalosome (CSN), an evolutionarily conserved regulator of the Cullin-based ubiquitin ligases that act in the proteasome pathway, has been found associated with diverse debilitating syndromes, suggesting that CSN may be involved in regulation of dendritic arborization. However, the mechanism of this control, if it exists, is unknown. To address whether the CSN pathway plays a role in dendrites, we used a simple and genetically tractable model, Drosophila larval peripheral nervous system. Our model study identified the COP9 signalosome as the key and multilayer regulator of dendritic arborization. CSN is responsible for shaping the entire dendritic tree through both stimulating and then repressing dendritic branching. We identified that CSN exerts its dualistic function via control of different Cullins. In particular, CSN stimulates dendritic branching through Cullin1, and inhibits it via control of Cullin3 function. We also identified that Cullin1 acts in neurons with the substrate-specific F-box protein Slimb to target the Cubitus interruptus protein for degradation.     

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.     

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 proteasome regulatory particle subunit Rpn6 is required for Drosophila development and interacts physically with signalosome subunit Alien/CSN2

The eukaryotic 26S proteasome plays a central role in ubiquitin-dependent intracellular protein metabolism. The multimeric holoenzyme is composed of two major subcomplexes, known as the 20S proteolytic core particle and the 19S regulatory particle (RP). The RP can be further dissected into two multisubunit complexes, the lid and the base complex. The lid complex shares striking similarities with another multiprotein complex, the COP9 signalosome. Several subunits of both complexes contain the characteristic PCI domain, a structural motif important for complex assembly. The COP9 signalosome was shown to act as a versatile regulator in numerous pathways. To help define the molecular interactions of the signalosome during Drosophila development, we performed a yeast two-hybrid screen to identify proteins that physically interact with subunit 2 of the complex, namely Alien/CSN2. Here, we report that Drosophila Rpn6, a non-ATPase subunit of the RP lid complex, interacts with Alien/CSN2 via its PCI domain. The temporal and spatial expression patterns of Rpn6 and alien/CSN2 overlap on a large scale during development providing additional evidence for their interaction in vivo. Analyses of an Rpn6 P element insertion mutant and newly generated Rpn6 alleles reveal that Rpn6 is essential for Drosophila development.     

Association of the mammalian proto-oncoprotein Int-6 with the three protein complexes eIF3, COP9 signalosome and 26S proteasome

The mammalian Int-6 protein has been characterized as a subunit of the eIF3 translation initiation factor and also as a transforming protein when its C-terminal part is deleted. It includes a protein domain, which also exists in various subunits of eIF3, of the 26S proteasome and of the COP9 signalosome (CSN). By performing a two-hybrid screen with Int-6 as bait, we have isolated subunits belonging to all three complexes, namely eIF3-p110, Rpt4, CSN3 and CSN6. The results of transient expression experiments in COS7 cells confirmed the interaction of Int-6 with Rpt4, CSN3 and CSN6, but also showed that Int-6 is able to bind another subunit of the CSN: CSN7a. Immunoprecipitation experiments performed with the endogenous proteins showed that Int-6 binds the entire CSN, but in low amount, and also that Int-6 is associated with the 26S proteasome. Taken together these results show that the Int-6 protein can bind the three complexes with various efficiencies, possibly exerting a regulatory activity in both protein translation and degradation.     

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     

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.