COP9 complex homolog subunit 5: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - COP9 complex homolog subunit 5

Synonyms -

Cytological map position - 89D1--2

Function - signaling enzyme

Keywords - protein degradation, oogenesis, axonogenesis, eye

Symbol - CSN5

FlyBase ID: FBgn0027053

Genetic map position - 3-

Classification - signalosome subunit 5, Jab1/MPN domain metalloenzyme motif

Cellular location - cytoplasmic

NCBI links: Precomputed BLAST | Entrez Gene | UniGene |

Recent literature
Kim, K., Yoon, J., Yim, J. and Kim, H. J. (2015). Deneddylase 1 regulates Deneddylase activity of the Cop9 signalosome in Drosophila melanogaster. Insect Sci [Epub ahead of print]. PubMed ID: 26332639
NEDD8 conjugation of Cullin has an important role in ubiquitin-mediated protein degradation. The COP9 signalosome, of which CSN5 is the major catalytic subunit, is a major Cullin deneddylase. Another deneddylase, Deneddylase 1, has also been shown to process the Nedd8 precursor. In Drosophila, the DEN1 mutants do not have increased levels of Cullin neddylation, but instead show a significant decrease in neddylated Cullin. This characteristic decrease in neddylated Cullins in the DEN1null background can be rescued by UAS-dDEN1WT overexpression but not by overexpression of mature NEDD8, indicating that this phenotype is distinct from the NEDD8-processing function of DEN1. This study examined the role of DEN1-CSN interaction in regulating Cullin neddylation. Overexpression of DEN1 in a CSN5hypo background slightly reduced unneddylated Cullin levels. The CSN5, DEN1 double mutation partially rescues the premature lethality associated with the CSN5 single mutation. These results suggest that DEN1 regulates Cullin neddylation by suppressing CSN deneddylase activity.

Lu, T., Wang, S., Gao, Y., Mao, Y., Yang, Z., Liu, L., Song, X., Ni, J. and Xie, T. (2015). COP9-Hedgehog axis regulates the function of the germline stem cell progeny differentiation niche in the Drosophila ovary. Development 142: 4242-4252. PubMed ID: 26672093
It has been proposed that escort cells (ECs) form a differentiation niche to control germline stem cells (GSC) lineage specification extrinsically in the Drosophila ovary. However, it remains poorly understood how the maintenance and function of the differentiation niche are regulated at the molecular level. This study reveals a new role of COP9 in the differentiation niche to modulate autocrine Hedgehog (Hh) signaling, thereby promoting GSC lineage differentiation. COP9, which is a highly conserved protein complex composed of eight CSN subunits, catalyzes the removal of Nedd8 protein modification from target proteins. Genetic results demonstrate that all the COP9 components and the hh pathway components, including hh itself, are required in ECs to promote GSC progeny differentiation. Interestingly, COP9 is required in ECs to maintain Hh signaling activity, and activating Hh signaling in ECs can partially bypass the requirement for COP9 in GSC progeny differentiation. Finally, both COP9 and Hh signaling in ECs promote GSC progeny differentiation partly by preventing BMP signaling and maintaining cellular processes. Therefore, this study demonstrates that the COP9-Hh signaling axis operates in the differentiation niche to promote GSC progeny differentiation partly by maintaining EC cellular processes and preventing BMP signaling. This provides new insight into how the function of the differentiation niche is regulated at the molecular level.

The COP9 signalosome (CSN) is linked to signaling pathways and ubiquitin-dependent protein degradation in yeast, plant and mammalian cells, but its roles in Drosophila development are just beginning to be understood. During oogenesis, one subunit of the CSN [COP9 complex homolog subunit 5 (CSN5/JAB1)], is required for meiotic progression and for establishment of both the AP and DV axes of the Drosophila oocyte. CSN5 mutations block the accumulation of the Egfr ligand Gurken in the oocyte, interfering with axis formation. CSN5 mutations also cause the modification of Vasa, which is known to be required for Gurken translation. This CSN5 phenotype (defective axis formation, reduced Gurken accumulation and modification of Vasa) is very similar to the phenotype of the spindle-class genes that are required for the repair of meiotic recombination-induced DNA double-strand breaks. When these breaks are not repaired, a DNA damage checkpoint mediated by mei-41 is activated (Ghabrial, 1999). Accordingly, the CSN5 phenotype is suppressed by mutations in mei-41 or by mutations in mei-W68, which is required for double strand break formation. These results suggest that, like the spindle-class genes, CSN5 regulates axis formation by checkpoint-dependent, translational control of Gurken. They also reveal a link between DNA repair, axis formation and the COP9 signalosome, a protein complex that acts in multiple signaling pathways by regulating protein stability (Doronkin, 2002).

CSN5 is also involved in eye differention and in glial cell migration of the optic lobe. CSN5 is expressed in photoreceptor cells of the Drosophila compound eye (R cells) and accumulates in the terminus of the Drosophila axons in developing neuropil of the brain's optic lobe. Through the analysis of a unique set of missense mutations CSN5 has been shown to be required for R cells to induce lamina glial cell migration in the optic lobe. In CSN5 missense mutations, R1-R6 axon targeting is disrupted. Genetic analysis of protein null alleles further reveals that the COP9 signalosome is required at an earlier stage of development for R cell differentiation. Whether these two effects are independent, or represent a single function for CSN5, is as yet undetermined (Suh, 2002)

Investigation of CSN function has shown that it regulates protein stability in pathways leading to ubiquitination and degradation by the proteasome (reviewed by Seeger, 2001; Schwechheimer, 2001b; Kim, 2001). Degradation of regulatory proteins by the ubiquitin system plays a crucial role in many signaling pathways. Ubiquitin modifies target proteins to tag them for degradation by the proteosome. The congugation of ubiquitin -- itself a small protein -- to a substrate protein is accomplished by a set of ubiquitinating enzymes. Ubiquitin is first activated by a ubiquitin-activating enzyme (E1), and then ligated to the substate by a ubiquitin-conjugating enzyme (E2) assisted by a ubiquitin protein ligase (E3). Proteins that carry a chain of ubiquitin moleculeas are recognized by the 26S proteasome and rapidly degraded. There is an increasing body of evidence that two machineries, one the proteasome and the second the COP9 signalsome, cooperate in regulating the stability of important cellular proteins. Coprecipitation studies have revealed that COP9 signalsome associates with SCF, a multimeric ubiquitin ligase responsible for ubiquitiniation of regulatory proteins. SCF contains Skp1, Hrt1, substrate-binding F-box proteins and a RING finger domain protein of the cullin family as major components. Cullin (see Drosophila lin-19-like (also known as Cullin1) is modified by the covalent attachment of the ubiquitin-like protein Nedd3. The data suggest that the COP9 signalsome complex mediates cleavage of Nedd8-cullin conjugates thereby regulating SCF. Inhibition of the COP9 signalsome deneddylation activity by the alkylating agent NEM suggested that removal of Nedd8 is accomplished by cysteine protease activity. Sequence comparision studies indicate that CSN5 is the only subunit of the complex with homology to cysteine proteases. Moreover, deletion of the CSN5 orthologue in budding yeast leads to accumulation of modified cullin. CSN5 is therefore a strong candidate for being the deneddylase. Therefore the COP9 signalsome regulates the SCF complex which carries out ubiquitinization. The ubiqitinized protein is subsequently degraded by the 26S proteosome (Seeger, 2002 and references therein).

The Cop9 signalsome may in addition function in the phosphorylation of specific substrates, specifically p53 and c-Jun, thus targeting them to degradation by the ubiquitin system (Naumann, 1999; Bech-Otschir, 2001).

CSN5 mutations interfere with axis formation in Drosophila. Polarization of the anteroposterior (AP) axis of the Drosophila oocyte occurs early in oogenesis, while the presumptive oocyte is still in the germarium. The dorsoventral (DV) axis is set up much later and relies on transfer of the AP axis polarity from the oocyte to the somatic follicle cells at the posterior end of the oocyte. During stages 4-6 in wild-type egg chambers, grk RNA that is localized next to the nucleus at the posterior end of the oocyte is translated and signals through the Egfr pathway to establish the adjacent follicle cells as posterior. After these posterior follicle cells signal back to the oocyte, microtubule orientation in the oocyte is reversed, and the oocyte nucleus migrates along the microtubules to an anterior corner of the oocyte. During stages 8-9 this anterior corner is defined as dorsal by translation of grk RNA and activation of Egfr signaling in the overlying follicle cells. Thus, grk signaling is required for elaboration of the AP axis and establishment of the DV axis (Doronkin, 2002 and references therein).

Recent results have shown that establishment of both AP and DV axes also depends on the successful repair of DNA double strand breaks (DSBs) that are formed during meiotic recombination (Ghabrial, 1999). Meiotic prophase begins in early region 2a of the germarium, and both recombination and repair are probably completed before oocyte determination occurs in region 2b (Doronkin, 2002 and references therein).

Meiosis and axis establishment are related to each other because the accumulation of Grk protein in the oocyte cytoplasm depends on the successful completion of meiotic recombination (Ghabrial, 1999). Mutations in the spindle-class genes, spindle-B (spn-B), spindle-C (spn-C) and okra (okr), cause a delay in oocyte determination and a failure to accumulate Grk protein, leading to defects in AP and DV patterning in late oogenesis. spn-B and okr encode Drosophila homologs of the RAD51 and RAD54 genes from yeast that are required for DSB repair. Their effects on Grk appear to be mediated by a DNA damage checkpoint governed by Mei-41, a Drosophila member of the ATM/ATR family of kinases that are required for DNA damage and recombination checkpoints in yeast, worms and humans, as well as flies. Because they eliminate the checkpoint, mei-41 mutations suppress the effects of spn or okr mutations. The spn and okr mutations can also be suppressed by mutations in mei-W68, which encodes the Drosophila homolog of yeast gene SPO11, a gene required for the induction of DSBs during recombination These results indicate that the spn or okr patterning defects result from activation of a meiotic checkpoint in response to the presence of unrepaired DSBs (Doronkin, 2002 and references therein).

Like the spindle-class genes, CSN5 is required for the repair of recombination-induced DSBs during Drosophila oogenesis. The CSN5 protein (also known as Jab1), is a subunit of the eight protein COP9 signalosome complex (CSN) originally identified in plants and conserved from plant to mammalian cells (for reviews, see Seeger, 2001; Schwechheimer, 2001b; Bech-Otschir, 2002). As the genes for the CSN subunits were identified, a striking similarity was noticed between them and the eight subunits of the regulatory lid of the proteasome, suggesting a common ancestry and related function. This similarity was intriguing because examinations of CSN function have shown that it regulates protein stability in pathways leading to ubiquitination and degradation by the proteasome (Doronkin, 2002 and references therein).

The CSN has been implicated in many regulatory and signaling functions including activation of the Jun transcription factor, stabilization of nuclear hormone receptors and interactions with integrins. Most relevant here, the CSN or its subunits have been shown to regulate multiple steps in the mitotic cell cycle. For example, the CSN regulates the ubiquitination and degradation of the CDK inhibitor, p27kip1, and either a small, CSN5-containing subcomplex or CSN5 alone promotes p27kip1 nuclear export (Yang, 2002; Tomoda, 1999). In addition, a CSN-associated kinase activity promotes degradation of p53 (Bech-Otschir, 2001), thereby allowing cell cycle progression (Doronkin, 2002).

In Drosophila, CSN5 is essential for development (Freilich, 1999) and is required in photoreceptor cells to induce glial cell migration (Suh, 2002). Homozygous CSN5-mutant clones disrupt both the DV and AP axes of the oocyte as a result of decreased Grk protein. These effects on axis determination appear to be caused by activation of the meiotic recombination checkpoint (Doronkin, 2002).

Establishment of both AP and DV polarity requires expression of the TGF-alpha homolog Gurken in the oocyte and activation of the Egf receptor and its downstream effectors in the adjacent follicle cells. CSN5 is required in the germline for these critical signaling events. Several results tie CSN5 to Grk-Egfr signaling: (1) CSN5 mutations affected both axes as shown by DV defects in the eggshell, mislocalization of bcd and osk RNAs in both the oocyte and embryo, and mislocalization of dpp, rho and twi expression in the embryo; (2) CSN5 germline clones affect the expression of follicle cell reporters for Grk-Egfr signaling: slbo and the PZ6356 enhancer trap in the posterior follicle cells, kek expression in the dorsal anterior follicle cells; (3) CSN5 alleles show strong genetic interactions with grk alleles, and (4) Grk protein is reduced in CSN5 germline clones, starting in region 2a of the germarium but still evident in stage 10 egg chambers or in ovary extracts (Doronkin, 2002).

Previous studies have shown that the accumulation of Grk protein can be affected by activation of a meiotic checkpoint in response to the persistence of DNA double-strand breaks. Mutations in several genes that play a role in DNA repair (okra, spn-B, spn-C and spn-D) activate this meiotic checkpoint and disrupt axial patterning in the oocyte. There is a remarkable similarity between the CSN5-mutant phenotype and defects caused by mutations in these spindle-class genes. In both cases mutant females produced eggs with a variety of partially penetrant eggshell defects: mild or strongly ventralized, dorsalized, or small eggs or eggs with multiple dorsal appendages. Embryonic patterning is also disrupted, and both axes are affected. As had been seen in spindle-class mutants, the oocyte of some CSN5-mutant egg chambers is positioned laterally or at the anterior end, and some have defects in karyosome morphology. There is also a similar, strong reduction in Grk protein, with one intriguing difference. At early stages of oogenesis in CSN5 mutants, the level of Grk protein is always strongly reduced, both in germline clones of the strong CSN5L4032 allele and in hypomorphic combinations of CSN5L4032 with viable excision mutants. Although Grk is also strongly reduced in CSN5L4032 germline clones at later stages, it often appears to be present at higher levels than in the germarium. With the hypomorphic combinations, it is often difficult to detect any reduction in Grk protein at later stages. By contrast, in spn-B and spn-D mutants, Grk accumulates normally in early oogenesis but then declines and is often undetectable by stage 9-10. In okr mutants, the amount of Grk protein varies from one egg chamber to the next in a single ovariole, but a bias towards lower levels at early stages has not been reported. Thus, there seem to be three different patterns of Grk accumulation in these mutants. CSN5 mutants appear to cause a more immediate response of Grk to DNA damage than do spn-B and spn-D mutants (Doronkin, 2002).

Because of the similarities between the phenotypes and because at least two of the spindle-class genes, okr and spn-B, encode components of the RAD52 DNA repair pathway, it seems likely that CSN5 directly or indirectly regulates DSB repair. The fact that mei-41 and mei-W68 mutations can suppress the CSN5 phenotypes reinforces this conclusion. Kinases in the ATM/ATR subfamily, which includes Mei-41, play a central role in checkpoint-mediated responses to DNA damage. These checkpoint kinases are thought to act as sensors of DNA damage, becoming activated on binding damaged DNA. Phosphorylation of several downstream effectors, including the Chk1 (Drosophila homolog: Grapes) and Chk2 (Drosophila homolog: loki) kinases and p53, then restrains cell cycle progression until the DNA damage is repaired and the checkpoint kinases dissociate from the DNA. In Drosophila mei-41 mutants, the checkpoint cannot be activated, and oocytes with damaged DNA, such as those mutant for spindle-class genes, can proceed through oogenesis. Suppression of CSN5 phenotypes by mei-41 mutations demonstrate that the CSN5-mutant lesion acts upstream of the DNA damage checkpoint and suggest that DSBs arising during meiotic recombination cannot be efficiently repaired in CSN5-mutant cells (Doronkin, 2002).

Suppression by mei-W68 restricts the possible role of CSN5 further. mei-W68 encodes a topoisomerase II-like protein homologous to S. cerevisiae Spo11 and has been proposed to create the DSBs needed to initiate meiotic recombination. In flies mutant for mei-W68, DSBs are absent and meiotic recombination is eliminated. In double mutants of mei-W68 with either okr, spn-B or spn-C, Grk protein accumulation and eggshell patterning are normal and other spindle-class defects are suppressed. Heterozygosity for mei-W68 is sufficient to suppress hypomorphic CSN5-mutant phenotypes. Combination of this result with the mei-41 suppression result indicates that CSN5 acts in the recombination pathway to regulate the formation of DSBs or their successful repair (Doronkin, 2002).

vasa mutants show similar effects on axis determination and Grk protein accumulation as do spindle mutants and CSN5 germ-line clones. However, the vasa phenotypes are not suppressed by mei-41 or mei-W68 mutations, indicating that Vasa acts downstream of the meiotic checkpoint. Indeed, Vasa is one of the targets of Mei-41 activity since Vasa electrophoretic mobility is changed in spn-B mutants but restored in mei-41 spn-B double mutants (Ghabrial, 1999). Since Vasa protein binds to grk mRNA and is required for both its localization in the oocyte and its translation, it seems likely that the checkpoint effects on Grk accumulation are directly mediated by Vasa, although other Mei-41 targets cannot be excluded. The results of this study show effects of CSN5 mutants on Vasa mobility and are entirely consistent with the previous spn-B results, as would be expected if both types of mutants activate the same checkpoint (Doronkin, 2002).

It is proposed that in CSN5-mutant oocytes DSBs created by Mei-W68 during meiotic recombination are repaired more slowly than in wild type. Accumulation of unrepaired DNA breaks would then activate the mei-41-dependent checkpoint leading to a block in the progression of meiotic prophase. Since activated Mei-41 is an ATR-related kinase, it might modify Vasa directly or through downstream kinases such as Chk1 or Chk2. Modified Vasa would then prevent efficient Grk translation. Because CSN5 mutants are likely to affect the stability rather than the presence or absence of repair proteins, the DNA DSBs might be slowly repaired during the checkpoint-induced delay, thereby allowing cell cycle progression to resume. Delayed repair might explain why the early CSN5 effects on Grk expression are stronger than at later times. It might also explain why CSN5-mutant phenotypes are weaker and less penetrant than in okra and spn-B mutants, in which repair proteins are absent and DNA probably remains unrepaired (Doronkin, 2002).

How might CSN5 regulate DNA repair? Two mechanisms of CSN activity have been reported, and either might affect the activity or stability of proteins involved in DNA repair. In addition, since there is an excess of CSN5 relative to other CSN subunits in many cells (Yang, 2002), CSN5 might regulate DNA repair independent of the large CSN complex (Doronkin, 2002).

The best-documented mechanism for CSN activity works through regulation of the SCF (Skp1/cullin-1/F-box) ubiquitin ligases (Lyapina, 2001; Yang, 2002). This pathway is attractive here because SCF-dependent ubiquitination mediates the degradation of many cell-cycle regulators, including not only p27kip1, but also cyclins E, A and B, CDK inhibitor p21, E2F1, ß-catenin and IkappaBalpha. Recently, a connection has been made in C. elegans between the SCF complex and the regulation of meiosis (Nayak, 2002). Members of the Skp1-related (skr) gene family in C. elegans are required for the restraint of cell proliferation, progression through the pachytene stage of meiosis, and formation of bivalent chromosomes at diakinesis (Doronkin, 2002 and references therein).

The CSN regulates SCF activity by removing the ubiquitinlike protein Nedd8 from the cullin subunit of SCF (Lyapina, 2001). Nedd8/Rub1 is covalently attached to target proteins through an enzymatic cascade analogous to ubiquitination. It is ligated to all cullin family proteins, and so far cullins are the only known targets for neddylation. Nedd8 modification enhances the ubiquitinating activity of the SCF complex in vitro and is required in vivo for embryogenesis in both mice and nematodes. Since the CSN mediates cleavage of the Nedd8 conjugate, it can antagonize SCF-dependent protein degradation. For example CSN inhibits ubiquitination and degradation of p27kip1 in vitro and injection of the purified complex inhibited the G1-S transition in cultured cells (Doronkin, 2002).

Although this deneddylation activity of the CSN would explain the results in Drosophila, the kinase activity associated with the CSN might also be important. This kinase activity co-purifies with the CSN complex though it is uncertain whether it is intrinsic to one of the CSN subunits (Bech-Otschir, 2002). It phosphorylates and stabilizes the Jun transcription factor against proteasomal degradation (Musti, 1997). Conversely, it sensitizes p53 degradation by the SCF-ubiquitin pathway (Bech-Otschir, 2001) (Doronkin, 2002 and references therein).

Although the DNA repair-related targets of CSN5 or the CSN remain unclear, proteins encoded by the spindle-class genes or by mei-W68 are strong candidates. The deneddylation activity of the CSN might protect a DNA repair protein from SCF-dependent degradation. Alternatively, the kinase activity might promote Mei-W68 turnover, thereby limiting the production of DSBs. Further investigation may help to distinguish among these and other hypotheses and find direct targets for CSN5 in oogenesis (Doronkin, 2002).


cDNA clone length - 1198

Bases in 5' UTR - 48

Exons - 3

Bases in 3' UTR - 166


Amino Acids - 327

Structural Domains

COP9 signalosome (CSN) cleaves the ubiquitin-like protein Nedd8 from the Cul1 subunit of SCF ubiquitin ligases. The Jab1/MPN domain metalloenzyme (JAMM) motif in the Jab1/Csn5 subunit underlies CSN's Nedd8 isopeptidase activity. JAMM is found in proteins from archaea, bacteria, and eukaryotes, including the Rpn11 subunit of the 26S proteasome. Metal chelators and point mutations within JAMM abolish CSN-dependent cleavage of Nedd8 from Cul1, yet have little effect on CSN complex assembly. Optimal SCF activity in yeast and both viability and proper photoreceptor cell (R cell) development in Drosophila melanogaster requires an intact Csn5 JAMM domain. It is proposed that JAMM isopeptidases play important roles in a variety of physiological pathways (Cope, 2002).

COP9 complex homolog subunit 5: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 14 December 2002

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.