InteractiveFly: GeneBrief

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

BIOLOGICAL OVERVIEW

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, p27^kip1, and either a small, CSN5-containing subcomplex or CSN5 alone promotes p27^kip1 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 CSN5^L4032 allele and in hypomorphic combinations of CSN5^L4032 with viable excision mutants. Although Grk is also strongly reduced in CSN5^L4032 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 p27^kip1, 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 p27^kip1 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).

REGULATION

Protein Interactions

The COP9 signalosome is an essential multi-subunit repressor of light-regulated development in plants. It has also been identified in mammals. This complex is similar to the regulatory lid of the proteasome and eIF3 and several of its subunits are known to be involved in kinase signaling pathways. In order to reveal the developmental function of the COP9 signalosome in animals, Drosophila genes encoding eight subunits of the COP9 signalosome have been isolated. Co-immunoprecipitation and gel-filtration analysis shows that these proteins are components of the Drosophila COP9 signalosome. Yeast two-hybrid assays indicated that several of these proteins interact, some through the PCI domain. Disruption of the CSN5 subunit by either a P-element insertion or deletion of the gene causes lethality at the late larval or pupal stages. This lethality is probably a result of numerous pleiotropic effects. These results indicate that the COP9 signalosome is conserved in invertebrates and that it has an essential role in animal development (Freilich, 1999).

DEVELOPMENTAL BIOLOGY

Embryonic

Since most CSN5 homozygotes die during larval or pupal development (Freilich, 1999), it seemed likely that embryos receive a maternal contribution of CSN5 RNA or protein. In situ hybridization confirms this expectation, showing that CSN5 RNA accumulates in the nurse cells beginning in the germarium and continuing through most of oogenesis. During stage 10, CSN5 RNA is transferred to the oocyte along with the bulk of the nurse cell cytoplasm. In embryos, uniformly distributed maternal RNA is evident until gastrulation begins. The earliest zygotic expression is in an anterior stripe during cellular blastoderm. During gastrulation, zygotic expression becomes evident in the ventral furrow, the cephalic furrow, and both the anterior and posterior midgut invaginations (Doronkin, 2002).

The COP9 signalosome converts temporal hormone signaling to spatial restriction on neural competence

During development, neural competence is conferred and maintained by integrating spatial and temporal regulations. The Drosophila sensory bristles that detect mechanical and chemical stimulations are arranged in stereotypical positions. The anterior wing margin (AWM) is arrayed with neuron-innervated sensory bristles, while posterior wing margin (PWM) bristles are non-innervated. This study found that the COP9 signalosome (CSN; see CSN5) suppresses the neural competence of non-innervated bristles at the PWM. In CSN mutants, PWM bristles are transformed into neuron-innervated, which is attributed to sustained expression of the neural-determining factor Senseless (Sens). The CSN suppresses Sens through repression of the ecdysone signaling target gene broad (br) that encodes the BR-Z1 transcription factor to activate sens expression. Strikingly, CSN suppression of BR-Z1 is initiated at the prepupa-to-pupa transition, leading to Sens downregulation, and termination of the neural competence of PWM bristles. The role of ecdysone signaling to repress br after the prepupa-to-pupa transition is distinct from its conventional role in activation, and requires CSN deneddylating activity and multiple cullins, the major substrates of deneddylation. Several CSN subunits physically associate with ecdysone receptors to represses br at the transcriptional level. A model is proposed in which nuclear hormone receptors cooperate with the deneddylation machinery to temporally shutdown downstream target gene expression, conferring a spatial restriction on neural competence at the PWM (Huang, 2014: PubMed).

EFFECTS OF MUTATION

To enable an analysis of early embryonic requirements for CSN5, homozygous, CSN5-mutant germline clones were induced. These clones revealed requirements for CSN5 during oogenesis as well as embryogenesis. In ovarian germline clones the level of CSN5 mRNA is dramatically reduced, but still detectable, indicating that the P-element-induced allele, CSN5^L4032, is hypomorphic. Depending on the paternal allele, embryos derived from the germline clones show either a reduced amount of CSN5 RNA in the zygotic pattern or no detectable CSN5 RNA (Doronkin, 2002).

Flies carrying CSN5 germline clones lay eggs with a range of abnormal phenotypes that are affected by temperature. Flies grown at 25°C laid eggs with phenotypes closest to normal. The most frequent defects at 18°C are different from those at 29°C. At 18°C many of the defective eggs have fused dorsal appendages. At 29°C there is an increasing frequency of properly separated but short dorsal appendages. These results suggest that aberrations in patterning the follicular epithelium predominate at 18°C, while defects in follicle cell migration predominate at 29°C (Doronkin, 2002).

Because the eggshell phenotypes were only partially penetrant, it is possible that they are caused by somatic, rather than germline, CSN5 clones. To test this possibility, somatic clones were induced in the ovary by using the follicle cell driver E22c-GAL4 to induce expression of UAS-FLP. Under these conditions, there were no eggshell defects at any temperature, indicating that this requirement for CSN5 function is limited to the germline (Doronkin, 2002).

In addition to the eggshell defects, the viability of CSN5 mutants also depends on temperature. At 29°C the original P-element mutation is lethal during early development with fewer than 1% of the mutant larvae becoming prepupae. By contrast, at 18°C 90% of the mutant larvae pupariate and 1-2% escape as adults. Mobilization of the original P-element insertion confirms that it is responsible, not only for lethality, but also for the eggshell defects; precise excisions are viable and have normal dorsal appendages (Doronkin, 2002).

Some mutations that disrupt the DV patterning of the eggshell also affect the patterning of the embryo. To look for effects on the embryonic DV fate map, the expression of three zygotic genes was used as markers: decapentaplegic (dpp), rhomboid (rho) and twist (twi). dpp is expressed on the dorsal side of the embryo as well as its anterior and posterior ends. rho is expressed in two, eight-cell-wide ventrolateral domains and later also in a narrow stripe on the dorsal side of the embryo. twi, a marker for the mesoderm, is expressed ventrally in the embryo (Doronkin, 2002).

For all three of the markers, many of the CSN5-mutant embryos appear to be ventralized. In these embryos dpp expression on the dorsal side is reduced or absent. The dorsal rho stripe is reduced and the lateral stripes are moved dorsally. twi expression appears to expand dorsally about halfway around the embryo. Some embryos show stronger ventralization at their anterior or posterior ends. Infrequently, there were also embryos that appeared to be dorsalized (Doronkin, 2002).

To characterize CSN5 mutants further, the spatial localization of the RNAs were examined for two determinants of AP polarity, bicoid (bcd) and oskar (osk). The localization of bcd RNA to the anterior pole of the oocyte is crucial in the establishment of AP polarity. In CSN5 mutant oocytes and embryos, bcd mRNA is abnormally expressed in 10%-15% of oocytes. In these abnormal oocytes, the bcd mRNA is diffusely distributed and sometimes accumulates near the center of the oocyte. In mutant embryos, the bcd RNA often shifts toward the dorsal side of the embryo (Doronkin, 2002).

The posterior pole of the egg chamber is defined by the tight, posterior localization of osk RNA. Although most CSN5-mutant oocytes and embryos are nearly normal, osk RNA in 10%-15% of mutant oocytes and embryos is reduced or mislocalized. In the abnormal oocytes, the osk RNA is typically diffuse or concentrated in the center of the oocyte. Only small amounts were localized at the posterior pole. In the abnormal embryos only a small amount of osk RNA at the posterior pole remains. In these embryos the osk RNA appears to be shifted slightly dorsally from its normal position at the extreme posterior end (Doronkin, 2002).

Since the localization of osk and bcd RNAs depends on polarization of the microtubule lattice, a reporter for the motor protein kinesin was used to examine microtubule organization in CSN5 germline clones. Kinesin moves toward the plus ends of microtubules, and in stage 8-9 wild-type egg chambers kinesin-ß-gal localizes to the posterior of the oocyte. However, in some CSN5-mutant oocytes kinesin-ß-gal staining was diffuse or mislocalized (Doronkin, 2002).

In addition to its role in determining the AP axis, CSN5 may have a distinct role in pole cell development. In normal embryos, the pole cells form as a tight, contiguous cluster at the posterior end of the embryo. As gastrulation and germ band extension begin, somatic epithelial cells at the posterior end of the embryo form a shallow cup that will eventually become the posterior midgut invagination. The pole cells adhere to this cup and remain tightly clustered on its surface as they are conveyed over the dorsal side of the embryo and then into its interior. In CSN5-mutant embryos the number of pole cells is often reduced, as might be expected because of the inefficient localization of oskar RNA. In addition, the pole cells are occasionally found in a loose, non-contiguous group near, but not tightly associated with, the posterior end of the embryo. This is an unusual phenotype, not seen in other mutants that impair the formation of pole plasm. Thus, in addition to its role in oskar RNA localization, CSN5 may have a separate role in organizing the pole cell cluster (Doronkin, 2002).

Since CSN5 germline clones cause defects in both the AP and DV axes, it seemed possible that grk signaling is compromised. grk is unusual among axis-determining genes in being required for both axes. To assess the role of CSN5 in grk signaling, reporters for either the posterior or the dorsal Grk signal were used. In the absence of the posterior Grk signal, the posterior follicle cells appear to adopt the anterior follicle cell fate and express markers that are characteristic of the border cells. Two such markers, an enhancer trap called PZ6356 and a slbo-lacZ enhancer trap, were used to monitor whether CSN5 is required for the early Grk signal. For both markers, loss of CSN5 from the germline causes lacZ expression in the posterior follicle cells of many egg chambers, suggesting a reduction in Grk signaling. To monitor Egfr signaling to the dorsal follicle cells at stages 9 and 10, a kekkon (kek)-lacZ reporter construct was used. Because the kek gene acts downstream of the Egfr pathway in the follicle cells, it can serve as a sensitive indicator of grk activity coming from the oocyte. At 18°C kek expression is abnormal in about a third of CSN5-mutant egg chambers at stage 10 (but only 3%-4% at 25°C). In most of these egg chambers, expression in the dorsal anterior follicle cells over the oocyte was reduced or, rarely, absent. A small number of egg chambers show broader expression of kek in the follicle cells, probably reflecting the small number of dorsalized embryos arising from these mutant egg chambers. It is concluded that in most egg chambers both posterior and dorsal Grk signaling are impaired in CSN5-mutant germline clones (Doronkin, 2002).

Further evidence that CSN5 affects Grk signaling comes from testing for genetic interactions between CSN5 and either grk or Egfr. Females heterozygous for strong grk alleles lay eggs with fused or partially fused dorsal appendages. This dominant phenotype provides a sensitive background for detecting interactions. With the exception of a precise P-element excision, all CSN5 alleles show strong enhancement of the dominant grk phenotype. In addition, CSN5^L4032 weakly enhances the dominant eggshell phenotype of a loss of function Egfr allele, Egfr^f2 (Doronkin, 2002).

These results suggested that production of grk RNA or protein might be affected in CSN5 germline clones. In situ hybridization using a grk probe showed normal or nearly normal localization of grk RNA in most CSN5-mutant stage 10 oocytes. In some of these mutant oocytes the messenge was improperly localized, probably because the oocyte nucleus was no longer located at the dorsal corner of the oocyte. Interestingly, in these oocytes the 'dorsal' follicle cells are often columnar as though the nucleus had been properly localized at an earlier stage. A Northern blot showed nearly normal amounts of grk mRNA in ovaries carrying CSN5-mutant germline clones, consistent with the strong signals seen by in situ hybridization in most oocytes (Doronkin, 2002).

Immunostaining of egg chambers using anti-Grk antibodies showed a more extreme effect. Grk protein is strongly reduced in CSN5 mutants compared with controls, although the residual protein usually appears to be properly localized. This reduction was confirmed by western blot analysis. There were also a few cases of Grk protein mislocalization, sometimes being present all along the anterior end of the oocyte (Doronkin, 2002).

The reduction in Grk protein appeared to be most extreme at early stages in oogenesis. Grk expression begins in region 2a in wild-type germaria. The signal appears in several cells per cyst in regions 2a and 2b and then becomes concentrated in the oocyte cytoplasm by region 3. In viable, hypomorphic combinations such as CSN5^ex21/CSN5^L4032, Grk expression could not be detected in the germarium. With this combination Grk does become detectable from stage 2-3 onwards, suggesting that a reduction in CSN5 causes a delay in the beginning of Grk accumulation. Taken together these results show that the major effect of CSN5 mutations appears to be on grk RNA translation or on stability of the protein (Doronkin, 2002).

Because of the similarity between the CSN5 and spindle-class phenotypes, a connection between CSN5 and the meiotic checkpoint mediated by mei-41 was tested. The viable hypomorphic combination CSN5^ex21/CSN5^L4032 causes a reduction in Grk protein level, especially during the early stages of oogenesis. Five to fifteen percent of eggs laid by these transheterozygotes had fused dorsal appendages, indicating a partial reduction of Grk. When CSN5^ex21/CSN5^L4032 flies were also homozygous-mutant for mei-41, however, the normal Grk protein level was restored, and the eggshell phenotype was rescued (Doronkin, 2002).

Interestingly, checkpoint activation leads to modification of the Vasa protein, as shown by a slightly reduced mobility during SDS polyacrylamide gel electrophoresis. This result is relevant to the spindle-class and CSN5 phenotypes because Vasa regulates translation of Gurken and, as a consequence, axial patterning. This Vasa modification is checkpoint dependent since it is present in spn-B mutants but absent in mei-41 spn-B double mutants (Doronkin, 2002 and references therein).

A similar reduced mobility of Vasa protein is detected in CSN5 mutants. For viable CSN5 mutants there were two Vasa bands: one corresponding to Vasa from wild-type ovaries and a second with lower mobility. In stronger mutant combinations, most of the Vasa protein was modified, while in weaker combinations most Vasa had normal mobility. The shift in Vasa mobility was suppressed by mei-41 mutations. Interestingly, removal of one dose of mei-41 completely restores normal Vasa mobility for a weak CSN5 combination. For stronger CSN5 mutants, full restoration of Vasa mobility requires removal of both mei-41 genes (Doronkin, 2002).

The gene mei-W68 is required for the initiation of meiotic recombination in Drosophila ovaries and is likely to induce DNA double strand breaks (DSBs) as recombination begins (McKim, 1998). Mutations in mei-W68 rescue spindle-class defects, including Grk protein accumulation, eggshell morphology and Vasa modification. These results suggested that since DSBs are not formed in the absence of mei-W68, DNA repair by the spindle-class genes is not required. A similar interaction is seen between mei-W68 and CSN5. Hetrerozygosity for mei-W68 is sufficient to suppress the phenotypes of both strong and weak CSN5 allelic combinations (Doronkin, 2002).

These data demonstrate that absence of CSN5 function during meiosis activates a DNA-damage checkpoint that is mediated by Mei-41. Because the reduction in DSBs in mei-W68 heterozygotes removes the requirement for CSN5, it is likely that CSN5 promotes DNA repair, as do the spindle-class genes (Doronkin, 2002).

CSN5 acts in photoreceptor cells to induce glial cells

The R1-R6 subclass of photoreceptor neurons connects to the first optic ganglion of the optic lobe, the lamina, and relies upon glial cells as intermediate targets. Conversely, R cells promote glial cell development, including migration of glial cells into the target region. The CSN5 subunit of the COP9 signalosome complex is expressed in R cells, accumulates in the developing optic lobe neuropil, and is required in R cells to induce lamina glial cell migration. In a set of CSN5 missense alleles, R1-R6 targeting is disrupted. Genetic analysis of protein null alleles also 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).

A single loss-of-function mutation, initially designated quo¹, was identified in an ethyl methane sulfonate (EMS) screen for defects in R cell connections. Two additional EMS-induced alleles were identified, quo² and quo³, from a collection of lethal mutations generated in the 89C/D region. quo¹ mutant animals die as larvae or pupae, while quo² and quo³ phenotypes are less severe with a lethal phase later during pupal development. In some cases, quo²/quo³ heterozygotes survived to adulthood.These animals are morphologically normal, but are uncoordinated and sluggish, and die a few days after eclosion (Suh, 2002).

In quo¹, quo², and quo³ mutant larvae, many R1-R6 axons fail to terminate in the lamina but instead project through this region into the medulla. This defect in target specificity was revealed using Ro-taulacZ, a marker for a subset of R1-R6 axons (i.e., R2-R5). In contrast to wild-type, approximately 60% of the R2-R5 neurons in quo¹ project into the medulla. Mistargeting in homozygous quo¹ mutants and in quo¹ in trans to a deficiency is indistinguishable. quo² and quo³ phenotypes are qualitatively similar to, but less severe than, quo¹ (Suh, 2002).

Meiotic recombination and deficiency mapping localized quo to the cytological region between bands 89C7 and 89D1. Complementation tests placed quo between the proximal breakpoint of Df(3R)RK6-3 and the distal breakpoint of Df(3R)Sbd104. A region of some 200 kb was isolated through positional cloning and approximately 20 kb of that region, defined by the two aforementioned breakpoints, demarcated the region containing the mutations. A panel of fragments was introduced into flies by P element-mediated transformation and tested for rescue of the quo connectivity defects and lethality. A genomic fragment of 6.6 kb rescues the mutant phenotype. A single open reading frame of 327 amino acids was identified within the fragment and cDNAs corresponding to it were isolated. A heat shock cDNA transgene rescued both lethality and the R cell targeting phenotype. NCBI Blast search results show that this cDNA encodes a protein 65% and 74% identical to Jun-activation-domain binding protein1 (JAB1) (Claret, 1996) and subunit 5 of the Arabidopsis COP9 signalosome (CSN5) (Kwok, 1998), respectively. Each of the three EMS alleles leads to specific amino acid substitutions in the JAB/MPN domain (as defined in the SMART program [EMBL]) in residues conserved between plant, fly, and human. The quo gene (and its alleles) is referred to as CSN5 and the Quo protein as JAB1/CSN5 (Suh, 2002).

To determine whether CSN5 is required in R cell afferents or the brain, genetic mosaic analyses were undertaken. CSN5¹ mutant patches in the retina, in otherwise heterozygous animals, were created by X-ray-induced mitotic recombination or by using FLP/FRT-mediated recombination driven by eye-specific expression of the FLP-recombinase. R1-R6 targeting in adult tissue was assessed using a marker specific for the projections of R1-R6 axons, Rh1-lacZ. Of 22 mosaic animals, 15 exhibited an R1-R6 mistargeting phenotype. These results were confirmed using genetic mosaic analyses in developing eye-brain complexes by generating eye tissue homozygous for CSN5¹ while the target was wild-type. These projections were assessed using a pan-R cell specific marker, mAb24B10. All animals of this genotype exhibited abnormal projections. These results and the finding that the CSN5 mutant phenotype is rescued by neuron-specific expression of a full-length cDNA are consistent with CSN5 acting in R cells to regulate R1-R6 targeting (Suh, 2002).

R cell differentiation and pattern formation are normal in CSN5¹, CSN5², and CSN5³ alleles. The targeting defect in CSN5¹, CSN5², and CSN5³ does not result from transformation of R1-R6 neurons into R7 and/or R8 neurons that normally project into the medulla. The mistargeted neurons continue to express the R1-R6-specific markers Rh1-LacZ and Ro-taulacZ. In the developing eye, Bar (expressed specifically in R1 and R6 neurons), Prospero (expressed in R7 neurons and nonneuronal cone cells), and Boss are expressed in patterns indistinguishable from wild-type. These results are consistent with plastic sections of homozygous adult mutant patches. Of some 704 ommatidia scored from 10 independent mosaic patches, the number, organization, and morphology of CSN5¹ mutant R cells in some 697 ommatidia were indistinguishable from wild-type. In the remaining seven ommatidia, a single R cell was missing. Small numbers of missing R cells have been observed in other connectivity mutants (Suh, 2002).

Developmental studies have indicated that R1-R6 axons initially recognize lamina glia as intermediate targets in the developing lamina prior to making connections to specific lamina neurons several days later. Based on morphological studies, it has been proposed that the establishment of precise patterns of R1-R6 projections relies on interactions between R1-R6 afferents and lamina glia cells. The notion that lamina glia, not lamina neuronal precursors, are intermediate targets for R1-R6 afferents is supported by phenotypic analyses of nonstop and hedgehog mutants. Defects in glial cell induction are not an indirect effect of R1-R6 mistargeting, since glial cells develop normally in other genetic backgrounds in which R1-R6 axons mistarget (Suh, 2002).

To assess whether CSN5 is required in R cells for the development of lamina glia, target development was examined using the glial-specific anti-Repo antibody, both in homozygous CSN5¹ and in genetic mosaics in which mutant R cell axons project into a wild-type brain. While R cell-dependent lamina precursor cell proliferation and neuronal differentiation occurs normally, lamina glial cell development is disrupted in CSN5¹ homozygotes. This mutant phenotype is also observed in wild-type targets innervated by CSN5¹ mutant R cells. In wild-type, three layers of glia (epithelial, marginal, and medulla glia) surround the lamina plexus where R1-R6 axons terminate. In CSN5¹ homozygotes and mosaic animals in which the eye is mutant and the target is wild-type, there is a marked reduction in lamina glial cell number (approximately 46% of that in wild-type), and the remaining cells form disorganized rows (Suh, 2002).

The effects of CSN5¹ on lamina epithelial and marginal glia were compared to the effects on medulla glia by analyzing mutant animals carrying enhancer trap markers for these two populations. The lamina glial cells were visualized using 1.3D2 enhancer trap line, and the medulla glial cells with MZ97. The number of marginal and epithelial cells are markedly reduced, and the cells are highly disorganized. In contrast, there is no appreciable effect on the number of medulla glia in CSN5¹ mutants. While the medulla glial cell layer was disrupted in some cases, in most preparations a continuous row of medulla glia forms (Suh, 2002).

Defects in lamina glial cell development could be due to a defect in glial cell migration or differentiation. To distinguish between these two possibilities, the distribution of glial cells was analyzed in the developing optic lobe using anti-repo antibody as a marker. Lamina glial cells are derived from groups of cells flanking the lamina plexus; glial cells generated in these regions then migrate into the target. In wild-type, glial cells express Repo as they migrate into the R cell projection field. In CSN5¹, there is an increase in the number of cells that accumulate at the lateral edges. In wild-type, there are 12.1 ± 2.0 cells, whereas in homozygous CSN5¹ mutants there are 21.9 ± 3.4 cells and in CSN5¹ mutant eyes projecting into a wild-type target, there are 19.0 ± 3.1 cells. This accumulation is consistent with a failure of many glial cells to migrate in from the margin and parallels the decrease in the number of marginal and epithelial glial cells in the lamina. Based on these observations, it is concluded that lamina glial cell migration is disrupted in CSN5¹mutants (Suh, 2002).

These defects in lamina glial cell development and in R1-R6 targeting are similar to those observed in nonstop mutants. In contrast to CSN5, nonstop is required in lamina glial cells, and not in R cell afferents, for glial cell differentiation. The R1-R6 hyperinnervation phenotype in nonstop is similar to that in CSN5 mutants. These data argue that CSN5 functions in R cells to promote normal cell migration and development of lamina glial cells. It remains possible that CSN5 plays a dual function in R cells being required in a non-cell-autonomous fashion to induce glial cell development and in a cell-autonomous function in R1-R6 growth cone targeting. This is thought unlikely, since R1-R6 mutant neurons in a small patch of mutant retinal tissue target to the lamina plexus in a fashion similar to wild-type, while mutant neurons show marked mistargeting when imbedded in large mutant patches (Suh, 2002).

The JAB1/CSN5 protein could act directly in R cell growth cones to mediate interaction with target cells or alternatively, it could act indirectly, for instance, in R cell nuclei to control gene expression required for R1-R6 targeting. To gain insight into how JAB1/CSN5 regulates R cell differentiation and interaction between R cell growth cones and lamina glial cell targets, the subcellular distribution of JAB1/CSN5 was determined. To assess the JAB1/CSN5 expression pattern in the developing visual system, a Myc-epitope tag was inserted into the C terminus of the CSN5 open reading frame in a genomic construct (myc-CSN5) and introduced into flies. Four independently generated transgenic lines were analyzed. In each case, the tagged transgene rescued the CSN5 lethality and connectivity phenotypes. This supports the view that the expression pattern observed using a Myc-tagged genomic construct accurately reflects the endogenous expression of JAB1/CSN5. Third instar eye-brain complexes of animals carrying the transgene were stained with anti-Myc antibody. Anti-Myc staining is predominantly localized to the cytoplasm throughout the developing eye disc. In the developing optic ganglia, anti-Myc staining is prominent in the lamina plexus, which at this stage in development largely comprises R cell axons and growth cones. Myc immunoreactivity also was enriched in the medulla neuropil. This is the region to which the R7 and R8 cells, as well as lamina and medulla neurons, send their axons. The expression of the Myc-tagged genomic construct in each of four transgenic lines was identical. The JAB1/CSN5 expression pattern is similar to other proteins previously shown to be required for signaling in growth cones (e.g., Dock and Pak) during R cell axon guidance and targeting (Suh, 2002).

Since the CSN5¹ homozygous phenotype is indistinguishable from the CSN5¹ over a deficiency, it seems likely that this allele is a strong loss-of-function or a null allele. Nevertheless, since all three CSN5 alleles are missense mutations, attempts were made to identify mutations that delete CSN5 coding sequence to unambiguously establish the null phenotype. Protein null mutations were generated by imprecise excision of a weak CSN5 mutant that carries a P element within the promotor region of the CSN5 locus. Six CSN5 protein null alleles were isolated. In contrast to the pupal lethality observed in animals carrying CSN5 missense mutations (i.e., CSN5¹, as well as CSN5² and CSN5³ ) or the late larval and pupal lethality of partial loss-of-function mutations in CSN5 (Freilich, 1999), protein null homozygotes die in early larval stages. A heat shock-driven CSN5 cDNA transgene rescues the lethality. One null allele, designated CSN5^N, was chosen for further phenotypic analysis. It is the result of a deletion of the entire open reading frame as demonstrated through both PCR analysis and Southern blots (Suh, 2002).

Since CSN5^N larvae do not survive to third instar, the eye phenotype was assessed in genetically mosaic animals. CSN5^N eye tissue was generated using FLP recombinase expressed under the control of the eyeless promoter (ey-FLP) to promote eye-specific FRT-mediated mitotic recombination between wild-type and CSN5^N chromosomes. Under these conditions, more than 50% of the cells within the eye disc are homozygous mutant. Plastic sections of adult mutant eye tissue stained with toluidine blue have revealed that both the position of R cells and their morphology are abnormal. Abnormalities in differentiating R cells were observed in CSN5^N clones in developing third instar eye discs. Expression of Elav, an early neuron-specific nuclear protein, and Chaoptin, a later appearing R cell surface protein, is greatly reduced in the mutant clones. However, Futsch, a neuronal microtubule-associated protein that appears at an early stage of R cell differentiation, and Boss, an early R8-specific protein, were expressed normally. Expression of other markers including Dachshund, Cubitus interruptus (Ci), Delta, and Atonal was similar to wild-type. Cell cycle progression in the eye disc appeared normal as assessed using BrdU incorporation. The lack of a cell cycle phenotype as assessed with BrdU incorporation is consistent with the observation that the size of homozygous CSN5^N mutant clones is similar to sister clones homozygous for the wild-type allele (Suh, 2002).

These data indicate that JAB1/CSN5 is critical for neuronal differentiation, while earlier fate specification, patterning, and cell cycle events appear to proceed largely as in wild-type. Cone cell development is also disrupted in CSN5^N since the expression of the cone cell markers, Cut and Sparkling, is reduced. Since R cells induce cone cells, it is possible that cone cell defects are due to an indirect effect of loss of JAB1/CSN5 in R cells (Suh, 2002).

In both plant and animal cells, JAB1/CSN5 assembles into a complex called the COP9 signalosome (Kwok, 1998; Freilich, 1999). While the function of this complex in animal cells is not understood, in plant cells, though highly pleiotropic, it plays a crucial role in regulating light-dependent gene expression. To address whether the CSN5 null phenotype reflects the loss of function of the COP9 signalosome function, the role of another subunit, CSN4, in eye development was assessed. A CSN4-null mutant strain that harbors a deletion of part of the CSN4 coding region was acquired. In Arabidopsis, all reported subunit mutations led to a loss of the COP9 signalosome (Karniol, 2000). Similarly, in both CSN4^N and CSN5^N fly mutants, an intact COP9 signalosome does not form. CSN4^N clones in the eye contain R cells with markedly abnormal cellular morphologies similar to R cells in CSN5^N mutant clones. CSN4^N and CSN5^N mutant phenotypes in developing eye discs are also similar, with a marked reduction of Elav and Chaoptin expression. These data are consistent with the CSN5 null mutant phenotype reflecting the loss of the COP9 signalosome function in the developing eye (Suh, 2002).

R cell axons engage in a complex dialogue with developing lamina cells. R cell growth cones produce Hedgehog and Spitz, which induce the final cell division of lamina neuronal precursors and the differentiation of lamina neurons, respectively. R cell axons have also been proposed to produce a third signal that induces lamina glial cell development, although the molecular identity of this signal remains unknown. Lamina glial cells, but not lamina neurons, are essential for R1-R6 targeting, since genetic ablation of lamina glial cells results in R1-R6 mistargeting to the medulla. JAB1/CSN5 protein is also required for the development of lamina glial cells and is enriched in regions in the optic lobe neuropil containing R cell growth cones. Since these glial cells act as intermediate targets in the lamina, the R1-R6 neurons mistarget to the medulla. In JAB1/CSN5 mutants, lamina glial cells accumulate at the lateral edges of the developing lamina neuropil where they contact R cell growth cones. The accumulation of lamina glia at these sites and the genetic requirement in R cells are consistent with the conclusion that JAB1/CSN5 is required for signaling between R cell growth cones and lamina glial cells (Suh, 2002).

Does JAB1/CSN5 play a direct role in regulating intercellular signaling pathways in R cell growth cones necessary for the induction of lamina glial cells? Recent studies in mammalian cells provide a precedent for JAB1/CSN5 function in contact-dependent intercellular interactions. JAB1/CSN5 has been shown to physically interact with LFA1, an integrin critical for the interaction between T cell receptors and antigen-presenting cells (Bianchi, 2000). Interaction between the T cell receptor and the MHC-peptide complex is converted into a high-affinity complex through the recruitment of additional adhesion molecules linking the cells together. This includes LFA1, an integrin that directly interacts with I-CAM, an Ig domain-containing protein expressed on the surface of the antigen-presenting cell. The costimulatory interaction between T cell and antigen-presenting cells creates a specialized contact area termed an ''immunological synapse.'' Transport and clustering of signaling components at the interface between these two cells leads to further amplification of intercellular signaling mediating the communication between them. It is attractive to envision that JAB1/CSN5 may contribute to the construction and/or maintenance of a structure supporting reciprocal interactions between the lamina glial cells and R cell axons (Suh, 2002).

Since the JAB1/CSN5 protein is also localized to R cell bodies, it is possible that it regulates lamina development indirectly, for instance, by controlling the expression of signals necessary for lamina glial cell induction. Indeed, studies in mammalian cells in culture and in plant cells provide a precedent for JAB1/CSN5 function in transcriptional regulation (reviewed in Chamovitz, 2001). Overexpression of mammalian JAB1/CSN5 stimulates Jun-dependent transcriptional activation (Claret, 1996), and the binding of JAB1/CSN5 to diverse proteins affects AP1 mediated transcription. Further support for the notion that JAB1/CSN5 regulates AP1-dependent transcription comes from studies in which the level of the COP9 signalosome, including CSN5, was elevated in cells by overexpressing another COP9 signalosome component, CSN2 (Naumann, 1999). These data raise the intriguing possibility that JAB1/CSN5 and the COP9 signalosome (more generally) regulate Jun-dependent intracellular pathways. However, since Jun mutations in the fly eye do not disrupt R1-R6 targeting, it is unlikely that the R cell innervation and glial cell induction defects reflect disruption of Jun regulation. Therefore, JAB1/CSN5 must be impinging on other signaling pathways to regulate R1-R6 targeting. Identifying these targets is an important future goal (Suh, 2002).

A role for JAB1/CSN5 in regulating gene expression is consistent with studies in Arabidopsis. Like true loss-of-function mutations in other subunits of the COP9 signalosome, antisense RNA inactivation of CSN5 in Arabidopsis derepresses a light-dependent developmental pathway in the dark. The COP9 signalosome facilitates the accumulation of the COP1 protein in the nucleus. COP1, probably acting as an E3 ubiquitin-ligase, in turn promotes degradation of the light-dependent transcriptional activator HY5. Hence, in the dark, the COP9 signalosome inhibits HY5-dependent transcription by regulating the cellular levels of HY5 itself. Through an unknown mechanism, light inactivates the COP9 signalosome function, export of COP1 from the nucleus, and stabilization of HY5. These findings raise the intriguing possibility that signaling events at the R cell growth cone could lead to changes in gene expression necessary for interactions between the growth cone and glia cell targets that are dependent upon the COP9 signalosome (Suh, 2002 and references therein).

A role for JAB1/CSN5 in photoreceptor cell differentiation was revealed through analysis of protein null alleles. Cells within null mutant clones express reduced levels of two neuronal differentiation markers: Elav, an RNA binding protein required for neuronal differentiation and Chaoptin, an R cell-specific, cell surface protein. Another neuronal marker, Futsch (a neuron-specific, microtubule-associated protein) is expressed normally. Consistent with a role for JAB1/CSN5 in differentiation, null mutant R cells survive into the adult and exhibit disrupted cellular morphologies. Interestingly, R cell neurons lacking Elav exhibit similar morphological defects. Defects are not restricted to R cell differentiation, however, since the expression of two cone cell homeodomain proteins (Cut and Sparkling) is also markedly reduced in CSN5 null mutants. Since R cells induce cone cell development, it remains unclear whether this reflects a role for CSN5 in cone cells. Early eye patterning genes such as Cubitis interruptus, dachshund, and atonal are expressed normally. Hence, JAB1/CSN5 plays a crucial role in R cell and cone differentiation but is largely dispensable for early patterning in the eye disc (Suh, 2002).

Since JAB1/CSN5 is found in Arabidopsis and Drosophila in two forms (a multisubunit complex, the COP9 signalosome, and in a smaller complex-independent form) (Freilich, 1999; Kwok, 1998), defects in eye development in CSN5 mutants may reflect the function of the JAB1/CSN5 monomer or the COP9 signalosome. To address this issue, the role of another component of the COP9 signalosome, CSN4, in eye development was analyzed. In Drosophila CSN4 mutants, the CSN complex does not form, but the JAB1/CSN5 monomer remains. Since CSN4 and CSN5 null phenotypes are indistinguishable, it is concluded that they reflect the essential function of the COP9 signalosome in eye development (Suh, 2002).

As opposed to the general R cell differentiation defects caused by the CSN4 and CSN5 null mutations, three different CSN5 missense mutations isolated in two independent screens specifically disrupt interactions between R cell afferents and lamina glial cells but do not lead to defects in R cell differentiation. These mutations are clustered within the JAB/MPN domain and lead to nonconservative substitutions in amino acids shared between plant, fly, and human JAB1/CSN5 (Suh, 2002).

What could be the biochemical basis for this specific connectivity phenotype? JAB1/CSN5 may be required for a single function; a reduction in its activity in the missense mutations may disrupt only the most sensitive process (e.g., lamina glial cell induction). Alternatively, JAB1/CSN5 may serve multiple functions. The missense mutations may selectively disrupt a subset of them that are dependent upon specific residues within the JAB/MPN domain, while the null mutation abolishes all JAB1/CSN5 functions. It is conceivable that these different functions are inherent to the different cellular forms of JAB1/CSN5 (e.g., complexed versus noncomplexed forms). In Arabidopsis, the CSN5 in the COP9 signalosome is primarily nuclear, while the CSN5 monomer is primarily cytoplasmic (Kwok, 1998). Therefore, the cytoplasmic localization of JAB1/CSN5 reported here could suggest that noncomplexed forms of JAB1/CSN5 are involved in R cell growth cone glial cell interactions, similar to the cytoplasmic form of JAB1/CSN5 that interacts with LFA1 (Suh, 2002).

Recent studies suggest that the multiple roles attributed to JAB1/CSN5 and the COP9 signalosome may reflect their function in regulating protein degradation. The COP9 signalosome positively regulates SCF complexes through the removal of Ned8 modifications from the cullin subunits (Lyapina, 2001), allowing for E3 activity. Mutations in the COP9 signalsome lead to the accumulation of multiple neddylated cullins in fission yeast (Zhou, 2001). Mutations in the Arabidopsis COP9 signalosome lead to the accumulation of ubiquitinated proteins (Peng, 2001). While the exact role of the COP9 signalosome in these processes is still obscure, it is proposed that the function of the COP9 signalosome and JAB1/CSN5 in R cells involves the regulation of protein degradation, probably through its interaction with specific E3 ligases. The specific missense mutations in CSN5¹, CSN5², and CSN5³ may selectively disrupt the interactions with a subset of neddylated substrates involved in controlling interactions between R cell growth cones and lamina glial cells. Further biochemical and genetic analyses are required to determine the relationship between the structure of JAB1/CSN5 and its function in R1-R6 targeting, glial cell induction, and R cell differentiation (Suh, 2002).

The COP9 signalosome promotes degradation of cyclin E during early Drosophila oogenesis

The COP9 signalosome (CSN) is an eight-subunit complex that regulates multiple signaling and cell cycle pathways. The CSN has been linked to the degradation of Cyclin E, which promotes the G1-S transition in the cell cycle and then is rapidly degraded by the ubiquitin-proteasome pathway. Using CSN4 and CSN5/Jab1 mutants, it has been shown that the CSN acts during Drosophila oogenesis to remove Nedd8 from Cullin1, a subunit of the SCF ubiquitin ligase. Overexpression of Cyclin E causes defects similar to those caused by mutations in CSN or SCFAgo subunits -- extra divisions or, in contrast, cell cycle arrest and polyploidy. Because the phenotypes are so similar and because CSN and Cyclin E mutations reciprocally suppress each other, Cyclin E appears to be the major target of the CSN during early oogenesis. Genetic interactions among CSN, SCF, and proteasome subunits further confirm CSN involvement in ubiquitin-mediated Cyclin E degradation (Doronkin, 2003).

To investigate cyst formation and differentiation in CSN5 germaria, wild-type and CSN5 ovaries were stained with anti-Hts antibody to highlight the fusome that connects all the cells of a cyst through the ring canals. Fusome development is essential for germline cyst formation. In CSN5 mutant germaria the fusome was often less branched, and sometimes there were more individual fusomes than in wild-type germaria. Furthermore, spherical spectrosomes (fusome precursors) are frequently found in more posterior regions of the germaria, probably indicating retarded fusome development. CSN5 null mutant clones eventually cease mitotic divisions and often become enormously polyploid. Along with the increase in DNA, these cells often contain oversized spectrosomes or structures similar to a fragmented fusome, indicating dramatic changes in fusome development. Some mutant clones lacked spectrosomes/fusomes. Usually, these clones were found a significant time after heat shock and were localized in ovarioles with no subsequent germline development. CSN4N mutant clones show similar undifferentiated cysts with enlarged cell nuclei and defective fusome development. These data suggest that the intact CSN complex is required for proper cyst divisions and fusome development. The polyploid, nondividing germ cells may be the germline stem cells. More than three of these large polyploid cells are never seen in a particular germarium, and they retain contact with somatic cells that probably correspond to the basal and terminal filament cells of normal germaria (Doronkin, 2003).

The Drosophila F box protein Archipelago (Ago) has been proposed to target Cyclin E for ubiquitin-mediated degradation in imaginal discs. The hypomorphic alleles ago¹, ago³, and ago⁴ were used to test for a similar role in Cyclin E degradation during oogenesis. Immunostaining shows that ago mutant clones marked by lack of GFP persistently accumulate Cyclin E at high levels. With one addition, these clones showed a similar range of phenotypes as those seen in CSN5 or CSN4 mutants or after overexpression of Cyclin E. Some mutant cysts had extra nurse cells and some had fewer than normal, and many were degenerating. Some cysts had been arrested after the stem cell division and some of these single-cell cysts were polyploid. Prominent in the ago clones was a phenotype that had not been previously noticed. Cyclin E accumulation in ago clones correlates with significantly DAPI-bright regions in nurse cell nuclei. Because these regions are likely to include heterochromatic sequences that are usually underreplicated during endoreplication, their enlargement may indicate a more complete replication of both heterochromatic and euchromatic sequences in ago clones. Although enlarged heterochromatin-rich regions are occasionally seen in CSN5 mutants and after Cyclin E overexpression, this phenotype is stronger in ago mutants, possibly suggesting a more specific role for ago in regulation of late replication (Doronkin, 2003).

In addition to the similar phenotypes between ago mutants and CSN, Nedd8, or cullin1 mutants, dominant interactions were found between ago and CSN mutants. CSN5/ago and CSN4/ago double heterozygotes show familiar ovarian defects: extra cystocyte divisions, fewer divisions but higher ploidy, and apoptotic egg chambers. These defects are very similar to the CSN5 mutant phenotype and to defects in oogenesis induced by Cyclin E overexpression. In addition, these double heterozygotes have enlarged heterochromatic regions in nurse cell nuclei, suggesting mutual CSN-ago control of late replication (Doronkin, 2003).

The regulatory lid of the proteasome is an eight-subunit complex that is closely related to the CSN. It appears to be necessary for the removal of ubiquitin side chains from the target protein as it is fed into the barrel of the proteasome for proteolysis. A mutation in the RPN6 subunit of the regulatory lid was tested for genetic interactions in oogenesis with CSN4 and CSN5 mutations. Both double heterozygotes show a strong interaction and the full range of CSN5-like ovarian defects, including apoptosis, incorrect number of mitotic divisions, and fusions of neighboring egg chambers (Doronkin, 2003).

The effect of the CSN on the activity of the SCF complex has been controversial. Although Nedd8 modification of Cullin1 stimulates SCF activity, the opposite process, deneddylation, has also been shown to be important for SCF function and cell cycle progression. For example, point mutations in the JAMM domain of the S. cerevisiae CSN5 homolog Rri abolish its deneddylation activity and enhance the growth defect shown by ts alleles of SCF genes. These results have led to the proposal that repeated cycles of neddylation and deneddylation are required for the sustained activity of the SCF. However, a recent gain-of-function analysis suggests that deneddylation by the CSN inhibits degradation of the SCF target p27^kip1 (Doronkin, 2003).

The results of this study strongly support the idea that deneddylation of Cullin1 by the CSN is necessary for activity of the SCF complex. CSN mutations have the same, not opposite, effects on oogenesis as do Nedd8, cullin1, or ago mutations. CSN5 and CSN4 mutations also interact dominantly with cullin1 and ago mutations, further suggesting that the CSN works along with the SCF to promote Cyclin E degradation. These requirements for the CSN appear to demand its deneddylase activity, because the CSN5^quo2 mutation, with a single amino acid substitution in the metalloprotease domain, behaves similarly to a CSN5 null (Doronkin, 2003).

Cycles of neddylation and deneddylation might control the association of an F box protein with an E3 ubiquitin ligase core complex or the association of a ubiquitin-loaded E2-conjugating enzyme with the E3 complex. Neddylation might also affect Cullin1 stability as suggested by the Cullin1 accumulation that is seen in CSN5 mutants and its reduction in Nedd8 mutants (Doronkin, 2003).

Conjugation of Nedd8 to cullins may regulate not only their activity, but also their subcellular distribution. Shuttling between the nucleus and cytoplasm has been proposed as a regulatory mechanism for E3 ubiquitin ligases when the target protein is ubiquitinated in the nucleus. The results showing that in CSN mutants, Nedd8-modified Cullin1 accumulates in the cytoplasm suggest that neddylation may be one way to regulate shuttling. Neddylation might favor nuclear export of Cullin1, and nuclear CSN would be required to remove Nedd8 and prevent export. Alternatively, neddylation might prevent Cullin1 nuclear import, and recycling of SCF into the nucleus would require cytoplasmic CSN. On either model, the CSN would be an important regulator of SCF activity. For example, modulation of SCF nuclear shuttling might affect the timing of Cyclin E degradation and entry into S phase of the cell cycle (Doronkin, 2003).

One of the important results of the current work is the demonstration that the CSN regulates the cell cycle in ovaries primarily through the turnover of Cyclin E. The apparent perdurance and gradual dilution of wild-type CSN5 protein in genetically null germline clones shows that reduced Cyclin E degradation affects both cell division and DNA replication. Slight reductions cause an extra division of the cystocytes. In contrast, continuous or strong accumulation of Cyclin E in null CSN5 mutants is able to reduce or stop cell divisions though often allowing endoreplication to continue. This switch from overproliferation to inhibition of cell divisions is sometimes visible in a single CSN5 mutant ovariole as the wild-type CSN5 protein is diluted by stem cell divisions. These observations support the view that different Cyclin E levels can lead to distinct and sometimes opposite effects (Doronkin, 2003).

Mutations in Drosophila ago, the C. elegans gene cul1, or the F box-encoding lin23 have been shown to cause increased cell proliferation, suggesting a critical role for SCF in regulating cell divisions. Extra cell division is found to be a frequent phenotype produced by mutations in CSN5, CSN4, cullin1, ago, or by overexpression of Cyclin E. However, SCF and CSN mutations have also been shown to cause the opposite effect on the cell cycle. In null mutant clones of cullin1 or Nedd8, cell proliferation in Drosophila eye discs is arrested. Similarly, loss of CSN5, CSN4, Cullin1, or ago inhibits and finally stops cell proliferation and often leads to enlarged nuclei. The abundance of Cyclin E and giant polyploid nuclei are also present in mice that are mutant for cul1 (Doronkin, 2003).

Elevated levels of Cyclin E that may give cells a proliferative advantage are found in many human tumors. In many of these tumors the Cyclin E gene itself is amplified. However, among breast and ovarian cancer cell lines that overexpress Cyclin E protein without amplification, several lines have mutations in hCDC4, the human homolog of archipelago, suggesting that SCF^[hcdc4] acts to suppress tumor formation. The results suggest that the CSN might have a similar effect (Doronkin, 2003).

In summary, these genetic and functional relationships between the CSN, the SCF, and the proteasome link these complexes in the regulation of Cyclin E degradation during normal development. When either the CSN or SCF are disrupted, the periodic degradation of Cyclin E is prevented, and cell cycle deregulation ensues (Doronkin, 2003).

CSN maintains the germline cellular microenvironment and controls the level of stem cell genes via distinct CRLs in testes of Drosophila melanogaster

Stem cells and their daughters are often associated with and depend on cues from their cellular microenvironment. In Drosophila testes, each Germline Stem Cell (GSC) contacts apical hub cells and is enclosed by cytoplasmic extensions from two Cyst Stem Cells (CySCs). Each GSC daughter becomes enclosed by cytoplasmic extensions from two CySC daughters, called cyst cells. CySC fate depends on an Unpaired (Upd) signal from the hub cells, which activates the Janus Kinase and Signal Transducer and Activator of Transcription (Jak/STAT) pathway in the stem cells. Germline enclosure depends on Epidermal Growth Factor (EGF) signals from the germline to the somatic support cells. Expression of RNA-hairpins against subunits of the COnstitutively Photomorphogenic-9- (COP9-) signalosome (CSN) in somatic support cells disrupted germline enclosure. Furthermore, CSN-depleted somatic support cells in the CySC position next to the hub had reduced levels of the Jak/STAT effectors Zinc finger homeotic-1 (Zfh-1) and Chronologically inappropriate morphogenesis (Chinmo). Knockdown of CSN in the somatic support cells does not disrupt EGF and Upd signal transduction as downstream signal transducers, phosphorylated STAT (pSTAT) and phosphorylated Mitogen Activated Protein Kinase (pMAPK), were still localized to the somatic support cell nuclei. The CSN modifies fully formed Cullin RING ubiquitin ligase (CRL) complexes to regulate selective proteolysis. Reducing cullin2 (cul2) from the somatic support cells disrupted germline enclosure, while reducing cullin1 (cul1) from the somatic support cells led to a low level of Chinmo. It is proposed that different CRLs enable the responses of somatic support cells to Upd and EGF (Qian, 2014).

EVOLUTIONARY HOMOLOGS

The COP9 signalosome complex in plants

The Constitutive Photomorphogenic9 (COP9) complex is a nuclear localized, multisubunit protein complex essential for repression of light-mediated development in Arabidopsis. Mutations that abolish the complex result in constitutive photomorphogenic development in darkness and pleiotropic developmental defects in both light and darkness. Two apparently redundant genes, AJH1 and AJH2, encode a subunit of the COP9 complex. Both AJH1 and AJH2 share high amino acid sequence identity (62 and 63%, respectively) with JAB1, a specific mammalian coactivator of AP-1 transcription. The proteins encoded by these two genes are present in both complex and monomeric forms, whereas complex formation is in part mediated by the direct interaction with FUSCA6. In addition, the stability of the monomeric AJH proteins requires functional COP1 and DEETIOLATED1 loci. Together with the fact that the previously known subunit FUSCA6 is an Arabidopsis homolog of human GPS1, a negative regulator of AP-1 transcription, these data suggest that the COP9 complex may contain both negative and positive regulators of transcription. Therefore, the COP9 complex may achieve its pleiotropic effects on Arabidopsis development by modulating activities of transcription factors in response to environmental stimuli (Kwok, 1998).

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. 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 (Peng, 2001).

The COP9 signalosome (CSN) is an evolutionarily conserved multiprotein complex that mediates the repression of photomorphogenesis in the dark in Arabidopsis through the degradation of transcription factors such as HY5 and HYH. CSN-mediated HY5 and HYH degradation also requires the activity of the putative E3 ubiquitin ligase (E3) component COP1 and the E2-conjugating enzyme variant COP10. CSN is also required for auxin responses mediated by the SCF-type E3 SCF(TIR1). To determine whether Arabidopsis CSN is required for E3-mediated processes in a more general manner, plants were generated with reduced E3 function by suppressing AtRBX1, an essential core subunit of SCF-type E3s. AtRBX1 transgenic plants share multiple phenotypes with CSN reduced-function plants, such as morphological defects and reduced responses to auxin, jasmonic acid, and cold stress, suggesting that CSN is required for multiple AtRBX1-mediated processes. Furthermore, mutants with defects in AXR1, a protein that had been described only as a regulator of SCF(TIR1) function, also is required for other E3-mediated processes and for the COP1/COP10/CSN-mediated repression of photomorphogenesis in the dark. It is concluded that CSN and AXR1 are of general importance for different pathways that are controlled by E3-mediated protein degradation (Schweichheimer, 2002).

The COP9 signalosome complex in Neurospora

The COP9 signalosome (CSN) promotes the function of SCF-type cullin-based ubiquitin ligase complexes in vivo. Paradoxically, removal of the Nedd8 modification of cullins by CSN inhibits the ubiquitin ligase activity of SCF complexes in vitro. Ubiquitination-mediated degradation of the Neurospora circadian clock protein Frequency (Frq) is critical for clock function. Ubiquitination of Frq requires FWD-1, the substrate-recruiting subunit of an SCF complex. Disruption of a subunit of CSN (csn-2) impairs the degradation of Frq and compromises its normal circadian expression. A Frq-independent oscillator drives conidiation in the csn-2 mutant, resulting in a 2-d conidiation rhythm that persists in constant darkness (DD), constant light (LL), light-to-dark (LD) transitions, and temperature cycles. Strikingly, the levels of FWD-1 are drastically reduced in csn-2 mutant, explaining the impaired degradation of Frq. Reduction of FWD-1 levels in the mutant requires its F-box, suggesting that its degradation is due to autoubiquitination. In addition, SKP-1 and CUL-1 of the SCF^FWD-1 complex are also unstable in the mutant. Therefore, these results establish an important role of CSN in the circadian clock of Neurospora. These findings also reconcile the CSN paradox and suggest that a major function of CSN is to maintain the stability of SCF ubiquitin ligases in vivo (He, 2005).

The COP9 signalosome complex in yeast

The function of the fission yeast cullins Pcu1p and Pcu4p requires modification by the ubiquitin-related peptide Ned8p. The COP9/signalosome (CSN) has been shown to regulate Ned8p modification of Pcu1p. Disruption of caa1/csn1, which encodes subunit 1 of the putative S. pombe CSN, results in accumulation of Pcu1p exclusively in the modified form. However, it remained unclear whether this reflects global control of all cullins by the entire CSN complex. Multiple CSN subunits control Ned8p modification of Pcu3p, another fission yeast cullin, which, in complex with the RING domain protein Pip1p, forms a ubiquitin ligase that functions in cellular stress response. Pcu3p is modified by Ned8p on Lys 729 and accumulates exclusively in the neddylated form in cells lacking the CSN subunits 1, 3, 4, and 5. These CSN subunits co-elute with Pcu3p in gel filtration fractions corresponding to approximately 550 kDa and specifically bind both native and Ned8p-modified Pcu3p in vivo. While CSN does not influence the subcellular localization of Pcu3p, Pcu3p-associated in vitro ubiquitin ligase activity is stimulated in the absence of CSN. Taken together, these data suggest that CSN is a global regulator of Ned8p modification of multiple cullins and potentially other proteins involved in cellular regulation (Zhou, 2001).

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. Csn1 and Csn2 have been identified 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. Null mutations of csn4 and csn5 have been characterized; both genes are required for removal of Nedd8 from the S. pombe cullin Pcu1 and their protein products associate with Csn1 and Csn2. However, neither csn4 nor csn5 null mutants share the csn1 and csn2 mutant phenotypes. The 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 (Mundt, 2002).

The COP9/signalosome (CSN) is known to remove the stimulatory NEDD8 modification from cullins. The activity of the fission yeast cullins Pcu1p and Pcu3p is dramatically stimulated when retrieved from csn mutants but inhibited by purified CSN. This inhibition is independent of cullin deneddylation but mediated by the CSN-associated deubiquitylating enzyme Ubp12p, which forms a complex with Pcu3p in a CSN-dependent manner. In ubp12 mutants, as in csn mutants, Pcu3p activity is stimulated. CSN is required for efficient targeting of Ubp12p to the nucleus, where both cullins reside. Finally, the CSN/Ubp12p pathway maintains the stability of the Pcu1p-associated substrate-specific adaptor protein Pop1p. It is proposed that CSN/Ubp12p-mediated deubiquitylation creates an environment for the safe de novo assembly of cullin complexes by counteracting the autocatalytic destruction of adaptor proteins (Zhou, 2003).

CSN-5 in C. elegans

The GLH proteins belong to a family of four germline RNA helicases in Caenorhabditis elegans. These putative ATP-dependent enzymes localize to the P granules, which are nonmembranous complexes of protein and RNA exclusively found in the cytoplasm of all C. elegans germ cells and germ cell precursors. To determine what proteins the GLHs bind, C. elegans cDNA libraries were screened by the yeast two-hybrid method, using GLHs as bait. Three interacting proteins, CSN-5, KGB-1, and ZYX-1, were identified and further characterized. GST pull-down assays independently established that these proteins bind GLHs. CSN-5 is closely related to the subunit 5 protein of COP9 signalosomes, conserved multiprotein complexes of plants and animals. RNA interference (RNAi) with csn-5 results in sterile worms with small gonads and no oocytes, a defect essentially identical to that produced by RNAi with a combination of glh-1 and glh-4. KGB-1 is a putative JNK MAP kinase that GLHs bind. A kgb-1 deletion strain has a temperature-sensitive, sterile phenotype characterized by the absence of mature oocytes and the presence of trapped, immature oocytes that have undergone endoreplication. ZYX-1 is a LIM domain protein most like vertebrate Zyxin, a cytoskeletal adaptor protein. In C. elegans, while zyx-1 appears to be a single copy gene, neither RNAi depletion nor a zyx-1 deletion strain results in an obvious phenotype. These three conserved proteins are the first members in each of their families reported to associate with germline helicases. Similar to the loss of GLH-1 and GLH-4, loss of either CSN-5 or KGB-1 causes oogenesis to cease, but does not affect the initial assembly of P granules (Smith, 2002).

The GLHs (germline RNA helicases), homologs of Drosophila Vasa, are constitutive components of the germline-specific P granules in the nematode C. elegans and are essential for fertility, yet how GLH proteins are regulated remains unknown. KGB-1 and CSN-5 are both GLH binding partners, previously identified by two-hybrid interactions. KGB-1 is a MAP kinase in the Jun N-terminal kinase (JNK) subfamily, whereas CSN-5 is a subunit of the COP9 signalosome. Intriguingly, although loss of either KGB-1 or CSN-5 results in sterility, their phenotypes are strikingly different. Whereas csn-5 RNA interference (RNAi) results in under-proliferated germlines, similar to glh-1/glh-4(RNAi), the kgb-1(um3) loss-of-function mutant exhibits germline over-proliferation. When kgb-1(um3) mutants are compared with wild-type C. elegans, GLH-1 protein levels are as much as 6-fold elevated and the organization of GLH-1 in P granules is grossly disrupted. A series of additional in vivo and in vitro tests indicates that KGB-1 and CSN-5 regulate GLH-1 levels, with GLH-1 targeted for proteosomal degradation by KGB-1 and stabilized by CSN-5. It is proposed the 'good cop: bad cop' team of CSN-5 and KGB-1 imposes a balance on GLH-1 levels, resulting in germline homeostasis. In addition, both KGB-1 and CSN-5 bind Vasa, a Drosophila germ granule component; therefore, similar regulatory mechanisms might be conserved from worms to flies (Orsborn, 2007).

The COP9 signalosome activates c-Jun

The basic region-leucine zipper transcription factor c-Jun regulates gene expression and cell function. It participates in the formation of homo- or hetero-dimeric complexes that specifically bind to DNA sequences called activating protein 1 (AP-1) sites. The stability and activity of c-Jun is regulated by phosphorylation within the N-terminal activation domain. Mitogen- and stress-activated c-Jun N-terminal kinases (JNKs) were previously the only described enzymes phosphorylating c-Jun at the N terminus in vivo. A JNK-independent activation of c-Jun in vivo directed by the constitutive photomorphogenesis (COP9) signalosome has been demonstrated. The overexpression of signalosome subunit 2 (Sgn2), a subunit of the COP9 signalosome, leads to de novo assembly of the complex with a partial incorporation of the overexpressed subunit. The de novo formation of COP9 signalosome parallels an increase of c-Jun protein resulting in elevated AP-1 transcriptional activity. The c-Jun activation caused by Sgn2 overexpression is independent of JNK and mitogen-activated protein kinase kinase 4. The data demonstrate the existence of a novel COP9 signalosome-directed c-Jun activation pathway (Naumann, 1999).

The COP9 signalosome interacts with an E3 ubiquitin ligase and thereby regulates protein degradation

The COP9 signalosome is an evolutionary conserved multiprotein complex of unknown function that acts as a negative regulator of photomorphogenic seedling development in Arabidopsis. Plants with reduced COP9 signalosome levels had decreased auxin response similar to loss-of-function mutants of the E3 ubiquitin ligase SCFTIR1. The COP9 signalosome and SCFTIR1 interact in vivo and the COP9 signalosome was required for efficient degradation of PSIAA6, a candidate substrate of SCFTIR1. Thus, the COP9 signalosome may play an important role in mediating E3 ubiquitin ligase-mediated responses (Schwechheimer, 2001a).

Nucleotide excision repair (NER) is a major cellular defense against the carcinogenic effects of ultraviolet light from the sun. Mutational inactivation of NER proteins, like DDB and CSA, leads to hereditary diseases such as xeroderma pigmentosum (XP) and Cockayne syndrome (CS). DDB2 and CSA are each integrated into nearly identical complexes via interaction with DDB1. Both complexes contain cullin 4A and Roc1 and display ubiquitin ligase activity. They also contain the COP9 signalosome (CSN), a known regulator of cullin-based ubiquitin ligases. Strikingly, CSN differentially regulates ubiquitin ligase activity of the DDB2 and CSA complexes in response to UV irradiation. Knockdown of CSN with RNA interference leads to defects in NER. These results suggest that the distinct UV response of the DDB2 and CSA complexes is involved in diverse mechanisms of NER (Groisman, 2003).

The COP9 signalsome promotes cleavage of NEDD8 from the cullin component of ubiquitin ligases

SCF ubiquitin ligases control various processes by marking regulatory proteins for ubiquitin-dependent proteolysis. To illuminate how SCF complexes are regulated, proteins that interact with the human SCF component CUL1 were sought. The COP9 signalosome (CSN), a suppressor of plant photomorphogenesis, associates 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. It is proposed 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 (Lyapina, 2001).

Cullin proteins assemble a large number of RING E3 ubiquitin ligases and regulate various physiological processes. Covalent modification of cullins by the ubiquitin-like protein NEDD8 activates cullin ligases through an as yet undefined mechanism. p120(CAND1) selectively binds to unneddylated CUL1 and is dissociated by CUL1 neddylation. CAND1 formed a ternary complex with CUL1 and ROC1. CAND1 dissociates SKP1 from CUL1 and inhibits SCF ligase activity in vitro. Suppression of CAND1 in vivo increases the level of the CUL1-SKP1 complex. It is suggested that by restricting SKP1-CUL1 interaction, CAND1 regulated the assembly of productive SCF ubiquitin ligases, allowing a common CUL1-ROC core to be utilized by a large number of SKP1-F box-substrate subcomplexes (Liu, 2002).

The COP9 signalsome inhibits p27 degradation via deneddylation of the cullin component of SCF ubiquitin ligases

The proliferation of mammalian cells is under strict control, and the cyclin-dependent-kinase inhibitory protein p27Kip1 is an essential participant in this regulation both in vitro and in vivo. Although mutations in p27Kip1 are rarely found in human tumors, reduced expression of the protein correlates well with poor survival among patients with breast or colorectal carcinomas, suggesting that disruption of the p27Kip1 regulatory mechanisms contributes to neoplasia. The abundance of p27Kip1 in the cell is determined either at or after translation, for example as a result of phosphorylation by cyclinE/Cdk2 complexes, degradation by the ubiquitin/proteasome pathway, sequestration by unknown Myc-inducible proteins, binding to cyclinD/Cdk4 complexes, or inactivation by the viral E1A oncoprotein. A mouse 38K protein (p38) encoded by the Jab1 gene interacts specifically with p27Kip1. Overexpression of p38 in mammalian cells causes the translocation of p27Kip1 from the nucleus to the cytoplasm, decreasing the amount of p27Kip1 in the cell by accelerating its degradation. Ectopic expression of p38 in mouse fibroblasts partially overcomes p27Kip1-mediated arrest in the G1 phase of the cell cycle and markedly reduces their dependence on serum. These findings indicate that p38 functions as a negative regulator of p27Kip1 by promoting its degradation (Tomoda, 1999).

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. Evidence suggests that both cullin neddylation and deneddylation are highly dynamic, and that equilibrium for neddylation 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 impedes 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. It is concluded 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 (Yang, 2002).

COP9 signalosome-specific phosphorylation targets p53 to degradation by the ubiquitin system

In higher eukaryotic cells, the p53 protein is degraded by the ubiquitin-26S proteasome system mediated by Mdm2 or the human papilloma virus E6 protein. COP9 signalosome (CSN)-specific phosphorylation targets human p53 to ubiquitin-26S proteasome-dependent degradation. As visualized by electron microscopy, p53 binds with high affinity to the native CSN complex. p53 interacts via its N-terminus with CSN subunit 5/Jab1 as shown by far-western and pull-down assays. The CSN-specific phosphorylation sites map to the core domain of p53 including Thr155. A phosphorylated peptide, Deltap53(145-164), specifically inhibits CSN-mediated phosphorylation and p53 degradation. Curcumin, a CSN kinase inhibitor, blocks E6-dependent p53 degradation in reticulocyte lysates. Mutation of Thr155 to valine is sufficient to stabilize p53 against E6-dependent degradation in reticulocyte lysates and to reduce binding to Mdm2. The p53T155V mutant accumulates in both HeLa and HL 60 cells and exhibits a mutant conformation. The mutant protein induces the cyclin-dependent inhibitor p21. In HeLa and MCF-7 cells, inhibition of CSN kinase by curcumin or Deltap53(145-164) results in accumulation of endogenous p53 (Bech-Otschir, 2001).

Integrin LFA-1 interacts with JAB1/CSN5

Integrin adhesion receptors transduce signals that control complex cell functions which require the regulation of gene expression, such as proliferation, differentiation and survival. The integrin intracellular domain has no catalytic function, indicating that interaction with other transducing molecules is crucial for integrin-mediated signaling. A protein has been identified that interacts with the cytoplasmic domain of the beta2 subunit of the alphaL/beta2 integrin LFA-1. This protein is JAB1 (Jun activation domain-binding protein 1), a coactivator of the c-Jun transcription factor. JAB1 is present both in the nucleus and in the cytoplasm of cells and a fraction of JAB1 colocalizes with LFA-1 at the cell membrane. LFA-1 engagement is followed by an increase of the nuclear pool of JAB1, paralleled by enhanced binding of c-Jun-containing AP-1 complexes to their DNA consensus site and increased transactivation of an AP-1-dependent promoter. It is suggested that signaling through the LFA-1 integrin may affect c-Jun-driven transcription by regulating JAB1 nuclear localization. This represents a new pathway for integrin-dependent modulation of gene expression (Bianchi, 2000).

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Search PubMed for articles about Drosophila CSN5

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