Cullins are the major components of a series of multimeric ubiquitin ligases that control the degradation of a broad range of proteins. The ubiquitin-like protein, Nedd8, covalently modifies members of the Cullin family. Nedd8 modifies Cullin 1 (Cul1, also known as Lin-19-like or simply Lin-19) in Drosophila. In mutants of Drosophila Nedd8 and Cul1, levels of the signal transduction effectors, Cubitus interruptus (Ci) and Armadillo, and the cell cycle regulator, Cyclin E (CycE), are unusually high, suggesting that the Cul1-based multimeric SCF ubiquitin ligase complex requires Nedd8 modification for the degradation processes of Ci, Arm, and CycE in vivo. Two distinct degradation mechanisms modulating Ci stability in the developing eye disc are separated by the morphogenetic furrow (MF) in which retinal differentiation is initiated. In cells anterior to the MF, Ci proteolytic processing promoted by PKA requires the activity of the Nedd8-modified Cul1-based SCFSlimb complex. In posterior cells, Ci degradation is controlled by a mechanism that requires the activity of Cul3, another member of the Cullin family. This posterior Ci degradation mechanism, which partially requires Nedd8 modification, is activated by Hedgehog (Hh) signaling and is PKA-independent (Ou, 2002).
The Hh pathway controls growth and pattern formation in many developmental processes in both vertebrates and invertebrates. The Hh signal is transmitted through a receptor complex consisting of Patched (Ptc) and Smoothened (Smo). In the absence of Hh, Ptc inhibits Smo activity, and the effector Cubitus interruptus (Ci) is phosphorylated by PKA, leading to the proteolysis of Ci, which is converted into Ci75 with the C terminus truncated. Ci75 functions as a transcriptional repressor in the Hh signaling pathway. Upon binding to Ptc, Hh relieves Smo from its repression state. Activated Smo mediates signaling to prohibit proteolytic processing of Ci. The intact full-length Ci (CiFL) functions as a transcriptional activator for expression of target genes of the Hh pathway (Ou, 2002).
In Drosophila, Hh signaling functions in patterning the A/P compartments in developing tissues such as embryonic segments and wing and leg imaginal discs. In development of the eye imaginal disc, Hh signaling is a major driving force of the retinal differentiation wave, the morphogenetic furrow (MF), which is caused by transient constriction in cell apical surface. The MF progresses anteriorly from the posterior margin of the eye disc during the third instar larval and early pupal stages. Anterior to the advancing MF, cells are proliferating, whereas posterior cells differentiate sequentially into photoreceptor, cone, or pigment cells. Transduction of Hh signaling in the MF is revealed by the accumulation of CiFL, which activates expression of target genes such as dpp and atonal in the MF. The induced MF cells soon differentiate and produce Hh proteins for further induction of more anterior cells, thus making the MF move forward (Ou, 2002).
The effect of neddylation on a broad spectrum of E3 ligases remains largely unknown. To investigate the role of neddylation in protein degradation control during developmental processes, Nedd8 and Cul1 mutants were identified and analyzed in Drosophila. The results suggest that neddylation is required for Cul1-mediated protein downregulation of the signaling pathway effectors Ci and Armadillo (Arm) and the cell cycle regulator CycE. Using the developing eye disc as a model system to study the regulation of CiFL stability, it was found that there is mechanistic difference in controlling CiFL stability between anterior and posterior cells separated by the MF. Whereas the Cul1-based SCFSlimb complex controls CiFL stability in anterior cells, a Cul3-dependent protein degradation mechanism controls CiFL stability in posterior cells. The differences between these two protein degradation mechanisms were further investigated (Ou, 2002).
In anterior cells of developing discs, CiFL proteolytic processing requires the activity of the Nedd8-modified, Cul1-based SCFSlimb complex. This CiFL proteolytic processing is inhibited by Smo signaling and promoted by PKA phosphorylation on CiFL. The mechanism by which CiFL is proteolyzed from CiFL to Ci75 is not clear. It is proposed that Nedd8 modifies and activates SCFSlimb for Ci ubiquitination and then proteolysis, as evidenced by Cul1 modification by Nedd8 and CiFL accumulation in Nedd8, Cul1, and slimb mutants. Consistently, proteolysis of CiFL depends on 26S proteasome activity. However, ubiquitinated Ci is not detected in cells treated with 26S proteasome inhibitors (Ou, 2002).
In the Hh signaling pathway, it is not clear how Smo signaling prevents CiFL from proteolysis. According to double mutant analysis, Nedd8 could be downstream or parallel to Smo and PKA signaling. Thus, it is possible that Hh signaling prevents CiFL from proteolysis through downregulating the level of Nedd8-modified Cul1. However, no change in the level of Nedd8-modified Cul1 could be detected in cell extracts prepared from the eye discs with ectopic Hh expression. It is therefore inferred that Hh may affect CiFL proteolysis through a Nedd8-independent mechanism (Ou, 2002).
Two modes of Ci downregulation in Drosophila eye development are proposed. In the undifferentiated cells anterior to the MF, Ci is phosphorylated by PKA constantly and processed by an SCFSlimb-dependent mechanism to generate the repressor form of Ci75. Upon binding to Hh, cells in the MF transduce Smo signaling to prevent this proteolytic processing. Thus, the transcriptional activator CiFL is preserved for activation of downstream genes in the MF (Ou, 2002).
In the posterior cells that are undergoing differentiation, a novel mechanism controls Ci degradation. Mutant analyses suggest that this mechanism is comprised of Smo signaling, Nedd8 modification, and Cul3 activity. The effect of Smo signaling in promoting Ci degradation in the posterior cells is in contrast to its effect on the anterior cells, in which Smo signaling prohibits CiFL processing. In addition to Smo signaling, Nedd8 modification activity also participates in this posterior Ci degradation. Further Cul1 mutant analysis suggests that Cullin proteins other than Cul1 are likely involved in this posterior degradation mechanism. This hypothesis has led to the identification of Cul3 as one candidate functioning in Ci degradation. More surprisingly, Cul3 activity is very restricted; Cul3 controls Ci degradation in the posterior, but not anterior, cells of the eye disc. CiFL accumulation may have an impact on proper differentiation of the posterior cells. In Cul3 mutants, cone cell differentiation is affected, probably due to the accumulation of CiFL (Ou, 2002).
Furthermore, the Ci degradation process is also distinct in posterior cells; Ci degradation is independent of PKA phosphorylation and proteolytic processing to the short form Ci-75. Based on these results, it is proposed that Smo signaling, acting in concert with the Nedd8 pathway, activates a Cul3-based ubiquitin ligase to degrade Ci in a PKA-independent mechanism in posterior cells of the eye disc (Ou, 2002).
It is not clear how Nedd8 modifies Cul3 in flies. Strong genetic interaction is observed between Nedd8 and Cul3 during eye and antennal development, suggesting that Nedd8 may also regulate Cul3. However, depletion of Nedd8 activity affects only posterior cells abutting the MF, in contrast to depletion of Cul3 activity, which increases the CiFL level in all posterior clones, indicating that some Cul3 activity is Nedd8-independent. It is possible that a basal Cul3 activity for Ci degradation is further enhanced by Nedd8 modification near the MF in which accumulated Ci may require efficient degradation for cells to enter proper differentiation (Ou, 2002).
Different protein-protein interactions may result in a switch between two Ci degradation mechanisms in eye discs. Ci is known to interact with Cos2, Fu, and Su(fu) to comprise a protein complex that promotes Ci degradation. Cos2, a motor-like protein with a kinesin motif, is required for tethering Ci in the cytosolic compartment and Ci proteolytic processing in the Drosophila developing wing. Similarly, Fu, a serine/threonine kinase, is also required for Ci processing. However, in Su(fu) mutants, levels of both long and short forms of Ci are reduced, suggesting that Su(fu) plays an additional role in Ci protein stability. Interestingly, the role of Su(fu) in controlling Ci stability seems modulated by Hh signaling. The results in this study indicate that, in contrast to the effect of Hh signaling in the anterior cells, Hh signaling downregulates the Ci level in the posterior cells of the eye disc. It is possible that the Ci protein complex is modulated by the sweep of the MF, and this change requires Hh signaling to expose Ci to the Cul3-based protein degradation machinery. Alternatively, additional factors may be activated by the sweeping of the MF and be required for Hh signaling to induce Cul3 activity that leads to constitutive Ci degradation (Ou, 2002).
Nedd8, the ubiquitin-like protein that covalently modifies members of the Cullin family, is highly conserved from yeast to mammals. Several Nedd8 alleles have been identifed in Drosophila, including two null alleles Nedd8AN015 and Nedd8AN024. The Nedd8 null mutants were growth-arrested in the first-instar larval stage and died within several days without further growth. Mutant clones were generated to analyze Nedd8 loss-of-function phenotypes; in the adult flies very few Nedd8AN015 mutant cells are identified, while in control experiments, large Nedd8+ clones are frequently recovered. Nedd8 mutant clones of small size, however, are present in the developing discs, suggesting that Nedd8 mutant cells are defective in proliferation and survival (Ou, 2002).
To study the relationship between Nedd8 and the F-box protein Slimb-mediated protein degradation, the protein stability for substrates of Slimb was studied in Nedd8 mutant cells. Nedd8 mutant cells in developing wing discs accumulate high levels of full-length Ci (CiFL) and Arm proteins, phenotypes identical to those observed in the slimb mutants. In Drosophila embryonic development, the signaling pathway mediated by the NFkappaB homolog Dorsal is required for patterning the dorsoventral identity. Accumulation of pIkappaBa/Cactus inhibits Dorsal activation, leading to repression of the downstream target gene, twist, an effect that has been observed in slimb mutants. twist expression was examined in embryos laid by Nedd8AN015/Nedd8203 females in which Nedd8203 is a hypomorphic allele. In such embryos, the twist expression domain is reduced along the dorsoventral axis and often found missing in many cells, revealing a requirement for Nedd8 in Dorsal signaling (Ou, 2002).
The Drosophila eye imaginal disc is an excellent model system for developmental study. Cells are undifferentiated and dividing randomly anterior to the MF, and cells posterior to the MF are differentiating into different types of cells. Thus, Nedd8 phenotypes can be observed in cells of different differentiation states in a single eye disc. The Hh pathway is the major signaling pathway in eye development, and the protein level of its effector Ci is tightly regulated in Drosophila. These studies focused on how Nedd8 regulates the CiFL level in the Hh pathway and the effects of Ci upregulation on eye development. It was found that in the Nedd8 clones that located anterior to the MF, CiFL accumulates to a level identical to that in the MF cells that transduce the Hh signaling pathway. Accumulation of CiFL also exists in posterior mutant cells that locate proximally but not distally to the MF. CiFL accumulation in Nedd8 mutant cells is not caused by an increase in the ci transcription level, because expression of ci-lacZ that recapitulates endogenous ci expression remains constant in Nedd8 mutant cells, indicating that posttranscriptional defects resulted in CiFL accumulation (Ou, 2002).
Elevated CiFL levels causes anterior Nedd8 mutant cells to adopt MF fate precociously. Nedd8 mutant cells are constricted on their apical surface, as revealed by the intensified phalloidin staining, and express the Hh-target gene, dpp, as detected by the expression of dpp-lacZ reporter gene. Furthermore, the early photoreceptor marker, Atonal, is induced. These phenotypes are observed only in mutant cells abutting the MF anteriorly, suggesting that accumulated CiFL in Nedd8 mutant cells is able to respond to Hh signaling (Ou, 2002).
CiFL accumulation in Nedd8 cells results from a defect in the machinery controlling CiFL protein processing. Ci protein processing is known to depend on the phosphorylation status of CiFL by PKA. The level of CiFL is downregulated when PKA is constitutively activated by the expression of its catalytic subunit. Therefore, the functional relationship between PKA activity and Nedd8 modification was examined. When the UAS-C* transgene was driven by eq-GAL4 for misexpression in the equator region of the eye disc, as visualized by the coexpressed GFP, the level of CiFL in the equator region was reduced, consistent with the observations that PKA phosphorylates Ci and promotes Ci proteolysis. Nedd8 mutant clones were then generated in the equator region where PKA is constitutively activated. In Nedd8 clones that overlap the eq-GAL4 expression domain, CiFL accumulates to a high level, identical to the level in the Nedd8 clone located externally to the eq-GAL4 expression domain. These results indicate that CiFL downregulation by PKA activity requires Nedd8 activity, and the effect of the Nedd8 pathway on CiFL processing is unlikely to be mediated through modulation of PKA activity (Ou, 2002).
The finding that CiFL accumulates in posterior smo3 clones indicates that Smo signaling contributes to the downregulation of CiFL in the posterior cells of the eye disc. This effect is in contrast to the smo role in the MF, where smo is required for CiFL activation. CiFL accumulation was also observed in the posterior Nedd8 mutant clones located proximally to the MF. In the smo3 Nedd8 double mutant clones, the level of CiFL is further enhanced, even in clones located distally to the MF, whereas no CiFL accumulation is detected in Nedd8 or smo3 clones, suggesting that Nedd8 and Smo function partially redundantly to downregulate Ci stability in the posterior cells of the eye disc (Ou, 2002).
The involvement of Nedd8 in controlling CiFL levels in the posterior cells of the eye disc suggests that Cullin proteins other than Cul1 may be involved in the posterior mechanism to control Ci stability. Among the mammalian Cullin family, Cul3 shares with the Cul1-based SCF complex the substrate CycE. To test whether Cul3 affects CiFL degradation in the eye disc, the available Drosophila Cul3 mutants were analyzed. CiFL accumulates in Cul3 mutant clones located posterior to the MF, with a higher level in nondifferentiating cells that surround differentiating photoreceptor clusters. In contrast, no CiFL accumulation is detected in anterior Cul3 mutant clones, indicating that Cul3 controls CiFL protein stability only in the posterior cells of the eye disc. Ci accumulation in posterior Cul3 mutant cells is controlled at the posttranscriptional level because ci expression is normal, as revealed by in situ hybridization. These results show that the CiFL degradation machinery in the posterior cells of the eye disc requires a Cul-3-mediated degradation mechanism. Ci accumulation is also detected in Cul3 mutant cells located in the A/P boundary of the wing disc. The level of Arm in Cul3 mutant clones in wing discs and the level of CycE in Cul3 mutant clones in eye discs remain constant, suggesting that Cul3 activity is specific to Ci (Ou, 2002).
In contrast to the Cul1-based SCFSlimb complex that controls CiFL processing only in the anterior cells of the eye disc, the Cul3-mediated Ci degradation mechanism is specific to the posterior cells. These specific activities in controlling Ci protein stability are not caused by differential gene expression of Cul1 and Cul3 in the eye disc. Ubiquitous mRNA expression patterns of both Cul1 and Cul3, and ubiquitous Cul1 protein expression are found all along the eye disc, suggesting that control of specificity is mediated by mechanisms other than regulation of Cul1 and Cul3 expression (Ou, 2002).
PKA phosphorylation promotes CiFL processing, and plays a role in the Hh signaling pathway for Ci activation. The requirement of PKA in CiFL degradation in the posterior cells of the eye disc was examined; CiFL downregulation is not regulated by PKA activity. Proteolytic processing of CiFL to the short form Ci75 is not a prerequisite for complete degradation in the posterior cells, in contrast to the proteolytic processing of the phosphorylated CiFL to the short form Ci75 in the anterior cells. To sum up, the results suggest that in the posterior cells of the eye disc, CiFL is degraded constitutively, and this degradation process is independent of PKA phosphorylation (Ou, 2002).
Cullin-dependent ubiquitin ligases regulate a variety of cellular and developmental processes by recruiting specific proteins for ubiquitin-mediated degradation. Cullin proteins form a scaffold for two functional modules: a catalytic module comprised of a small RING domain protein Roc1/Rbx1 and a ubiquitin-conjugating enzyme (E2), and a substrate recruitment module containing one or more proteins that bind to and bring the substrate in proximity to the catalytic module. This study presents evidence that the three Drosophila Roc proteins are not functionally equivalent. Mutation of Roc1a causes lethality that cannot be rescued by expression of Roc1b or Roc2 by using the Roc1a promoter. Roc1a mutant cells hyperaccumulate Cubitus interruptus, a transcription factor that mediates Hedgehog signaling. This phenotype is not rescued by expression of Roc2 and only partially by expression of Roc1b. Targeted disruption of Roc1b causes male sterility that is partially rescued by expression of Roc1a by using the Roc1b promoter, but not by similar expression of Roc2. These data indicate that Roc proteins play nonredundant roles during development. Coimmunoprecipitation followed by Western or mass spectrometric analysis indicate that the three Roc proteins preferentially bind certain Cullins, providing a possible explanation for the distinct biological activities of each Drosophila Roc/Rbx (Donaldson, 2004).
One possible explanation for the inability of a given Roc protein to rescue the phenotype of a different Roc mutant is that each Roc protein may form a unique set of E3 ubiquitin ligase complexes by preferentially interacting with different Cullin family members. To test this, coimmunoprecipitation experiments were performed with Roc1agrf::FLAG-Roc transgenes. Lysates from control, nontransgenic (w1118) embryos or embryos expressing each of the FLAG-Roc transgenes were incubated with anti-FLAG-agarose and immunocomplexes were analyzed by Western blotting or mass spectrometry. Western analysis with a CUL-1 antibody showed that CUL-1 efficiently coprecipitates with FLAG-Roc1a. Relatively little, but still above-background, amounts of CUL-1 was present in immunocomplexes from FLAG-Roc1b or FLAG-Roc2 lysates. This result shows that whereas Roc1a, Roc1b, and Roc2 are each able to bind to CUL-1 when expressed from the Roc1a promoter, Roc1a does so much more efficiently. Immunocomplexes were analyzed from each of the FLAG-Roc transgenic lysates by mass spectrometry. Proteins from a Coomassie-stained polyacrylamide gel that migrated with the predicted molecular weight of the Cullins and that were present in one or more of the transgenic lines but absent from wild-type, nontransgenic lysate were excised and identified by tandem mass spectrometry. Using this approach, CUL-1 and CUL-2 was identified in Roc1a immunocomplexes, CUL-3 in Roc1b immunocomplexes, and CUL-5 in Roc2 immunocomplexes. Because weaker Cullin-Roc interactions may not permit the precipitation of enough Cullin protein to be visible on a Coomassie-stained gel, this technique does not rule out any particular Cullin-Roc interactions. However, the data do suggest that there is a preference for the formation of certain Cullin-Roc complexes (Donaldson, 2004).
The results indicate that there are significant differences in the biological roles of the three Drosophila Roc proteins and that these differences are not simply the result of distinct expression patterns during development. In all of the experimental paradigms, Roc1a and Roc1b could partially, but not completely, substitute for one another, whereas Roc2 showed no ability to substitute for either Roc1 paralogue. Results of coimmunoprecipitation experiments suggest that these differences are due to preferential interactions between Roc and Cullin family members. For example, CUL-1 seems to interact most strongly with Roc1a, suggesting that a majority of SCF (i.e., CUL-1) targets require Roc1a. However, it cannot be ruled out that Roc1b or Roc2 function within the context of an SCF complex, since both showed weak interactions with CUL-1. Indeed, Roc1b seems to be capable of participating in SCF-mediated ubiquitylation, because it was able to rescue the aberrant accumulation of Ci, a bona fide SCF target, when overexpressed (Donaldson, 2004).
Because the Drosophila Roc proteins share between 40% and 60% overall sequence identity, it is somewhat surprising that a higher degree of complementation was not observed in rescue assays. Most of the conservation is within the C-terminal 67 residues, which contains the catalytic RING domain. Roc1a and Roc1b share 76% identity and 88% similarity in this domain, whereas Roc1a and Roc2 are 45% identical and 59% similar. In the N-terminal regions, the sequence identity/similarity is lower (38%/50% between Roc1a and Roc1b; 41%/57% between Roc1a and Roc2). Deletion of the Rbx1 (Roc1) N-terminus prevents interaction with CUL-1 in 293T cells. The crystal structure of the SCF complex shows that the association between Rbx1 and the C-terminal portion of the CUL-1 protein (termed the Cullin homology domain or CHD) consists of two parts. First, the RING domain of Rbx1 packs into a V-shaped groove formed by the alpha/B and WH-B domains of CUL-1. Second, the Rbx1 N terminus threads into CUL-1 and makes a five-stranded intermolecular ß-sheet (four strands provided by CUL-1 and one by Rbx1). This intermolecular ß-sheet seems to provide the primary mechanism of Rbx1 recruitment. Together, these data implicate the N terminus of the Drosophila Roc proteins as the region responsible for mediating the differential binding to Cullins (Donaldson, 2004).
This study used the powerful genetic techniques of the fruit fly to assess how the RING domain subunit contributes to the function of Cullin-dependent ubiquitin ligases. The Drosophila Roc proteins have nonredundant roles during development, and these differences may be mediated by the formation of specific Cullin-Roc ligase complexes. The results are consistent with studies of mammalian Roc proteins showing that although both Rbx1 and mammalian Roc2 can associate with all Cullin proteins, these interactions, as well as the associated ligase activities of the different complexes, seem to show certain preferences. Because each Cullin family member may use a distinct mechanism to target nonoverlapping sets of proteins for ubiquitylation, preferential Cullin binding provides a sufficient, if not the only, explanation for the functional differences among the three Drosophila Roc proteins. Further experiments are needed identify which complexes exist in vivo and to determine exactly what mediates these specific Cullin-Roc interactions (Donaldson, 2004).
The final step in Hedgehog (Hh) signal transduction is post-translational regulation of the transcription factor, Cubitus interruptus (Ci). Ci resides in the cytoplasm in a latent form, where Hh regulates its processing into a transcriptional repressor or its nuclear access as a transcriptional activator. Levels of latent Ci are controlled by degradation, with different pathways activated in response to different levels of Hh. The roadkill (rdx) gene is expressed in response to Hh. The Rdx protein belongs to a conserved family of proteins that serve as substrate adaptors for -mediated ubiquitylation. Overexpression of rdx reduces Ci levels and decreases both transcriptional activation and repression mediated by Ci. Loss of rdx allows excessive accumulation of Ci. rdx manipulation in the eye revealed a novel role for Hh in the organization and survival of pigment and cone cells. These studies identify rdx as a limiting factor in a feedback loop that attenuates Hh responses through reducing levels of Ci. The existence of human orthologs for Rdx raises the possibility that this novel feedback loop also modulates Hh responses in humans (Kent, 2006; full text of article).
The rdx locus was identified by an enhancer trap with embryonic expression in a pattern suggesting Hh-regulation. When genomic DNA flanking the insertion was used to screen a Drosophila embryonic cDNA library, cDNAs were obtained that initiated near the enhancer trap insertion and spliced into a cluster of seven downstream exons. These cDNAs represented the predicted gene CG10235 spliced into the predicted gene CG9924. ESTs recovered by the BDGP identified four additional isoforms (CG9924 A-D), which differ in their 5' ends but which share the cluster of seven downstream exons with rdxE, and are designated rdxA-rdxD (Kent, 2006).
The A, C/D and E forms are predicted to encode proteins with unique and novel amino termini fused to a common C terminus. The B form lacks unique coding sequence and is predicted to initiate translation within exon 7. The 398 C-terminal residues encoded by exons 7-13 contains two conserved domains: a MATH (Meprin and TRAF homology) domain and a BTB (Broad/Tramtrack/Bric-a-brac) domain. These two protein interaction domains are found together in an evolutionarily conserved protein family where the BTB domain binds to Cul3, while the MATH domain recruits specific substrates to the Cul3-based E3 ubiquitin ligase complex for ubiquitylation and subsequent degradation (Kent, 2006).
rdxA, rdxE and the initial enhancer trap produced expression patterns that were indistinguishable from those of a probe common to all rdx forms. Maternally deposited rdx transcripts were detected in early embryos, but disappeared during mid-cleavage stages. The first zygotic transcripts appeared in pole cells. During cellularization of the blastoderm, rdx transcripts appeared in two broad stripes in the head, in seven narrower stripes along the segment primordium, and in a ring surrounding the pole cells. Seven additional stripes appeared during germ band extension, so that by stage 8, rdx was expressed in 14 evenly spaced ectodermal stripes characteristic of segment polarity genes. At this time, strong expression was seen in the anterior and posterior midgut primordia. During stage 9/10, expression appeared in a subset of neuroblasts. During stage 10 each segmental stripe split so that by stage 11, ectodermal expression consisted of two thin stripes corresponding to the anterior and posterior margins of the former stripe. At this time, strong expression was seen in the mesoderm. As germ band retraction began, expression faded from most of the ectoderm, but was retained in the salivary glands and in abdominal segment 9. After stage 14, rdx expression was detected only in the clypeolabrum, anal plate and salivary glands (Kent, 2006).
Thus, rdx encodes a protein belonging to a phylogenetically conserved protein family of substrate-specific adaptors for Cullin3-based ubiquitin E3 ligases. rdx loss-of-function and gain-of-function studies suggest that rdx has at least two substrates: a regulator of early embryonic mitoses and the Hh regulated transcription factor Ci155. The data support a model where Rdx regulates the Hh-dependent degradation of Ci by acting as the adaptor that presents Ci to the Cul3-based E3 ubiquitin ligase. Because rdx is expressed in response to Hh, rdx is involved in a novel regulatory loop that attenuates Hh responses through reducing levels of Ci. In the wing, this feedback regulation of Ci by rdx plays a minor role, but in the eye it is essential for proper packing of ommatidia into a hexagonal array (Kent, 2006).
Hh is key regulator in human health. The haploinsufficiency of Ptc in humans and its activity as a morphogen in the spinal column argue that the level of Hh response is often crucial. Although there are differences in the Hh pathway between flies and vertebrates, many regulatory mechanisms are conserved. In particular, Gli2 and Gli3 are regulated much like Ci, becoming repressors or activators, depending on levels of Hh. The Rdx ortholog SPOP lies in 17q21.33, a chromosomal region that has been linked with ovarian cancer and cervical immature teratoma. Future studies will determine whether the Rdx orthologs SPOP or LOC339745 modulate Gli levels and Hh-mediated responses, and even contribute to cancer (Kent, 2006).
In both insects and mammals, spermatids eliminate their bulk cytoplasm as they undergo terminal differentiation. In Drosophila, this process of dramatic cellular remodeling requires apoptotic proteins, including caspases. To gain further insight into the regulation of caspases, a large collection of sterile male flies was screened for mutants that block effector caspase activation at the onset of spermatid individualization. This study describes the identification and characterization of a testis-specific, Cullin-3-dependent ubiquitin ligase complex that is required for caspase activation in spermatids. Mutations in either a testis-specific isoform of Cullin-3 (Cul3Testis), the small RING protein Roc1b, or a Drosophila orthologue of the mammalian BTB-Kelch protein Klhl10 all reduce or eliminate effector caspase activation in spermatids. Importantly, all three genes encode proteins that can physically interact to form a ubiquitin ligase complex. Roc1b binds to the catalytic core of Cullin-3, and Klhl10 binds specifically to a unique testis-specific N-terminal Cullin-3 (TeNC) domain of Cul3Testis that is required for activation of effector caspase in spermatids. Finally, the BIR domain region of the giant inhibitor of apoptosis-like protein dBruce is sufficient to bind to Klhl10, which is consistent with the idea that dBruce is a substrate for the Cullin-3-based E3-ligase complex. These findings reveal a novel role of Cullin-based ubiquitin ligases in caspase regulation (Arama, 2007; full text of article).
Caspases are executioners of apoptosis but also participate in a variety of vital cellular processes. This study has identified Soti, an inhibitor of the Cullin-3-based E3 ubiquitin ligase complex required for caspase activation during Drosophila spermatid terminal differentiation (individualization). Evidence is provided that the giant inhibitor of apoptosis-like protein dBruce is a target for the Cullin-3-based complex, and that Soti competes with dBruce for binding to Klhl10, the E3 substrate recruitment subunit. Soti is expressed in a subcellular gradient within spermatids and in turn promotes proper formation of a similar dBruce gradient. Consequently, caspase activation occurs in an inverse graded fashion, such that the regions of the developing spermatid that are the last to individualize experience the lowest levels of activated caspases. These findings elucidate how the spatial regulation of caspase activation can permit caspase-dependent differentiation while preventing full-blown apoptosis (Kaplan, 2010).
Programmed cell death is one of the most fundamental processes in biology. A morphologically distinct form of this active cellular suicide process, dubbed apoptosis, serves to eliminate unwanted and potentially dangerous cells during development and tissue homeostasis in virtually all multicellular organisms. Members of the caspase family of proteases are the central executioners of apoptosis. Caspases start off as inactive proenzymes and are activated upon proteolytic cleavage by other caspases. Apoptotic caspases can also participate in a variety of vital cellular processes, including differentiation, signaling, and cellular remodeling. However, the mechanisms that protect these cells against excessive caspase activation and undesirable death have remained obscure (Kaplan, 2010).
In both insects and mammals, spermatids eliminate their bulk cytoplasmic content as they undergo terminal differentiation. In Drosophila, an actin-based individualization complex (IC) slides caudally along a group of 64 interconnected spermatids, promoting their separation from each other and the removal of most of their cytoplasm and organelles into a membrane-bound sack called the cystic bulge (CB), which is eventually discarded as a waste bag (WB). This vital process, known as spermatid individualization, is reminiscent of apoptosis and requires apoptotic proteins including active caspases. However, the mechanisms that restrict caspase activation in spermatids, as opposed to their full-blown activation during apoptosis, are poorly understood (Kaplan, 2010).
The isolation of a Cullin-3-based E3 ubiquitin ligase complex required for caspase activation during spermatid individualization has been described (Arama, 2007). Ubiquitin E3 ligases tag cellular proteins with ubiquitin, thereby affecting protein localization, interaction, or turnover by the proteasome. The Cullin-RING ubiquitin ligases (CRLs) comprise the largest class of E3 enzymes, conserved from yeast to human. Cullin family proteins serve as scaffolds for two functional subunits: a catalytic module, composed of a small RING domain protein that recruits the ubiquitin-conjugating E2 enzyme, and an adaptor subunit which binds to the substrate and brings it within proximity to the catalytic module. In Cullin-3-based E3 ligase complexes, BTB-domain proteins interact with Cullin-3 via the eponymous domain, while they bind to substrates through additional protein-protein interaction domains, such as MATH or Kelch domains. A large body of evidence indicates that substrate specificity and the time of ubiquitination are determined by posttranslational modifications of the substrates and the large repertoire of the adaptor proteins. In addition, the Cullins themselves are subject to different types of posttranslational regulation. Most notably, they are activated by a covalent attachment of a ubiquitin-like protein Nedd8 (Kaplan, 2010).
This study has identified a small protein called Soti that specifically binds to Klhl10, the adaptor protein of a Cullin-3-based E3 ubiquitin ligase complex required for caspase activation during the nonapoptotic process of spermatid individualization. Soti acts as a pseudosubstrate inhibitor of this E3 complex and inactivation of Soti leads to elevated levels of active effector caspases and progressive severity of individualization defects in spermatids. Furthermore, the giant inhibitor of apoptosis (IAP)-like protein dBruce is targeted by this E3 complex, and this effect is antagonized by Soti. Finally, immunofluorescence studies reveal that Soti is expressed in a distal-to-proximal gradient, which promotes a similar distribution of dBruce in spermatids. Consequently, activation of caspases is restricted in both space and time, displaying a proximal-to-distal complementary gradient at the onset of individualization (Kaplan, 2010).
The current study provides insight into how some cells can utilize active caspases to promote vital cellular processes but still avoid unwanted death. According to this model, during early and advanced spermatid developmental stages, a gradient of Soti is generated, allowing graded activation of the Cullin-3-based E3 ubiquitin ligase complex in the opposite direction. This E3 complex then targets dBruce, promoting its distribution in a similar gradient as that of Soti. Subsequently, caspase activation occurs in a complementary gradient descending from proximal to distal. Since the removal of the cytoplasm and caspases also occurs in the direction of proximal to distal, the regions of the developing spermatid that are the last to individualize are also those that are the most protected against activated caspases. This setting ensures that each spermatidal domain encounters similar transient levels of activated caspases throughout the process of individualization (Kaplan, 2010).
The gradual regulation of caspase activity in spermatids is attributed to the outstanding length of Drosophila spermatids (a phenomenon called sperm gigantism). Spermatozoa of Drosophila melanogaster are about 1.9 mm long and other Drosophilids can produce sperm up to 58 mm long. Spermatids in Drosophila individualize over the course of 12 hr through a constant rate of proximal-to-distal individualization complex movement and clearance of the cytoplasmic content (including the active caspases) into a cystic bulge. Since it takes a few hours for the active effector caspasesto kill a cell, spermatids had to develop an efficient mechanism to prevent prolonged exposure of the more distal cellular regions to caspase activity. A gradient of a caspase inhibitor, descending from distal to proximal, is therefore an elegant mechanism to ensure a level of caspase activity that is sufficient to drive spermatid differentiation, yet not high enough to engage an apoptotic program (Kaplan, 2010).
Ubiquitination may target dBruce for either degradation or active redistribution. Because of technical limitations of the in vivo system, the biochemical analyses were performed in a heterologous system using truncated dBruce versions, and thus, we cannot completely rule out the possibility that at least some of the ubiquitinated dBruce is degraded by the proteasome. However, the genetic data support a model where dBruce may be redistributed by an active translocation mechanism, as hyperactivation of the Cullin-3-based complex, following Soti inactivation, leads to accumulation of dBruce at tail ends of spermatids and not to its elimination. This idea is also indirectly supported from the experiment in the eye system, showing that transgenic expression of the dBruce mini-gene enhanced the small eye phenotype caused by expression of Klhl10, suggesting that the Cullin-3-based complex does not target the dBruce mini-gene for degradation in this system. Consistent with this notion, accumulating evidence indicates that the ubiquitin 'code' on target proteins can be read by a large number of ubiquitinbinding proteins, which translate the ubiquitin code to specific cellular outputs, such as protein redistribution. Interestingly, a recent report suggests that Cullin-3-based polyubiquitination of caspase- 8 promotes its aggregation, which subsequently leads to processing and full activation of this protease. Furthermore, another Cullin-3-based ubiquitin ligase complex was shown to regulate the dynamic localization of the Aurora B kinase on mitotic chromosomes. Therefore, Cullin-3-based ubiquitin ligase complexes appear to promote also nondegradative ubiquitination and redistribution of proteins (Kaplan, 2010).
Cullin-RING ubiquitin ligases (CRLs) bind to substrates via adaptor proteins. However, adaptor proteins can also bind to pseudosubstrate inhibitors in a manner which is reminiscent of an E3-substrate-type interaction. Several lines of evidence strongly suggest that Soti is a pseudosubstrate inhibitor of the Cullin-3-based E3 ubiquitin ligase complex in spermatids. (1) The interaction between Klhl10 and Soti is an E3-substrate-type interaction. (2) Soti is not a substrate for this E3 complex. (3) dBruce polypeptides can outcompete with Soti for binding to Klhl10. Finally, Soti is a potent inhibitor of this E3 complex. Therefore, the mechanism of regulation by pseudosubstrates may represent a more common mechanism for modulation of CRL activity than has been previously appreciated (Kaplan, 2010).
Two alternative protein degradation pathways were recently described: N-terminal ubiquitination (NTU) and degradation 'by default'. Whereas the former promotes degradation of proteins by ubiquitination at N-terminal residues, the latter targets proteins for degradation by a ubiquitin-independent, 20S proteasome-dependent mechanism. Although these results cannot conclusively distinguish between these two pathways, two notable mechanistic traits of degradation 'by default' can be also attributed to Soti, including the targeting of intrinsically disordered proteins and their protection by binding to other proteins ('nannies'). Using the FoldIndex tool, Soti was predicted to be intrinsically disordered, while it is stabilized by attachment of a structured Myc-tag to its N terminus. Furthermore, Soti is highly unstable in the absence of its binding partner Klhl10, suggesting that Klhl10 functions as a 'nanny' for its own inhibitor. In conclusion, this study has uncovered a mechanism that restricts caspase activation during the vital process of spermatid individualization. This process appears to be conserved both anatomically and molecularly from Drosophila to mammals (reviewed in detail in Feinstein-Rotkopf and Arama, 2009). Moreover, several recent studies suggest that a similar Klhl10-Cul3 complex is essential for late spermatogenesis in mammals. Therefore, although the mammalian sperm is about 30 times shorter than in Drosophila, similar mechanisms (albeit scaled-down) for regulation of caspase activation may also exist during mammalian spermatogenesis. Further studies of the link between the ubiquitin pathway and apoptotic proteins during sperm differentiation in Drosophila may, therefore, provide new insights into the etiology of some forms of human infertility (Kaplan, 2010).
Cullin-RING ubiquitin ligases (CRLs), which comprise the largest class of E3 ligases, regulate diverse cellular processes by targeting numerous proteins. Conjugation of the ubiquitin-like protein Nedd8 with Cullin activates CRLs. Cullin-associated and neddylation-dissociated 1 (Cand1) is known to negatively regulate CRL activity by sequestering unneddylated Cullin1 (Cul1) in biochemical studies. However, genetic studies of Arabidopsis have shown that Cand1 is required for optimal CRL activity. To elucidate the regulation of CRLs by Cand1, a Cand1 mutant was analyzed in Drosophila. Loss of Cand1 causes accumulation of neddylated Cullin3 (Cul3) and stabilizes the Cul3 adaptor protein HIB. In addition, the Cand1 mutation stimulates protein degradation of Cubitus interruptus (Ci), suggesting that Cul3-RING ligase activity is enhanced by the loss of Cand1. However, the loss of Cand1 fails to repress the accumulation of Ci in Nedd8(AN015) or CSN5(null) mutant clones. Although Cand1 is able to bind both Cul1 and Cul3, mutation of Cand1 suppresses only the accumulation of Cul3 induced by the dAPP-BP1 mutation defective in the neddylation pathway, and this effect is attenuated by inhibition of proteasome function. Furthermore, overexpression of Cand1 stabilizes the Cul3 protein when the neddylation pathway is partially suppressed. These data indicate that Cand1 stabilizes unneddylated Cul3 by preventing proteasomal degradation. This study proposes that binding of Cand1 to unneddylated Cul3 causes a shift in the equilibrium away from the neddylation of Cul3 that is required for the degradation of substrate by CRLs, and protects unneddylated Cul3 from proteasomal degradation. Cand1 regulates Cul3-mediated E3 ligase activity not only by acting on the neddylation of Cul3, but also by controlling the stability of the adaptor protein and unneddylated Cul3 (Kim, 2010).
The neddylation pathway is highly conserved in many organisms, and the neddylation step is essential for Cullin-mediated E3 ubiquitin ligase activation. Cand1 is a highly conserved protein that binds to unneddylated Cullins and sequesters Cul1 from the CRL complex. It has been suggested that Cand1 inhibits CRL activity in vitro. However, studies from Arabidopsis have shown that loss of Cand1 leads to decreased CRL activity, indicating that Cand1 is required for efficient CRL function. Drosophila was used as a model system to elucidate this paradoxical effect of Cand1. First, it was found that loss of Cand1 increases the ratio of Nedd8 modified to unmodified Cul3 and the level of Cul3 adaptor HIB/rdx, causing enhanced degradation of CiFL, despite little effect on Cul1. Although Cand1 has been reported to negatively regulate CRL activity by binding to unneddylated Cul1 and dissociating the CRL complex in vitro, accumulations of neddylated Cullin and adaptor protein have never been observed in studies of Cand1 depletion. These provide a better understanding of the role of Cand1 in vivo, suggesting that the regulations of Cul3 neddylation and adaptor stability are important for Cand1 to control CRL activity. Unlike the results of Arabidopsis studies, in which Cand1 is required for optimal CRL activity, this study demonstrates that the Cand1 mutation of Drosophila stimulates the degradation of CiFL by enhancing Cul3-RING ligase activity. In addition, a novel insight is provided into the role of Cand1 by which Cand1 is involved in the stabilization of unneddylated Cul3. Evidence is presented that Cand1 protects unneddylated Cul3 from proteasomal degradation (Kim, 2010).
The absence of Cand1 increased the level of neddylated Cul3, and it suggests that Cand1 could inhibit the neddylation of Cul3. However, the overexpression of Cand1 had no effect on Cul3 neddylation. The amount of Cand1 seems to be sufficient to prevent Cul3 neddylation in the wild-type background. However, neddylation of Cul3 was decreased when Cand1 was expressed in the Cand1 mutant background, indicating that Cand1 can suppress Cul3 neddylation (Kim, 2010).
CiFL is processed by two different Cullins, Cul1 and Cul3, in the eye disc of Drosophila. In the posterior area of the eye imaginal disc, CiFL is degraded by Cul3-mediated E3 activity, where loss of Cand1 affects the stability of CiFL. Because it was observed that mutation of Cand1 decreases the level of CiFL, the levels of adaptor proteins of Cul1 and Cul3 were further investigated. It was found that the level of the Cul3 adaptor protein HIB/rdx is also increased in the Cand1 mutant, whereas the levels of Slimb, the F-box protein of the Cul1 RING ligase, remain constant. It suggests that Cand1 could regulate Cul3-based E3 ligase activity by suppressing the level of HIB/rdx. Several adaptor proteins are destabilized by autoubiquitination of CRL activity. CSN also maintains adaptor stability by deneddylating Cullin and recruiting deubiquitination enzymes. Interestingly, it has recently been observed that the CSN-associated deubiquitinating enzyme Ubp12 maintains the stability of the Cul3 adaptor, but not the F-box, Cul1 adaptor. This provides a possible clue that Cand1 may regulate the stability of HIB/rdx through deubiquitinating enzymes by working with CSN. Direct interaction of Cand1 with HIB/rdx suggests another possibility that Cand1 might suppress the level of HIB/rdx through a direct association with HIB/rdx. Taken together, the evidence presented in this study indicates that Cul3-dependent E3 ubiquitin ligase activity is increased by the loss of Cand1 function (Kim, 2010).
It has been suggested that Nedd8 covalent conjugation to Cullin causes instability of the Cullin protein. However, the current results show that the neddylated form of Cul3 has maintained protein stability in the Cand1 mutant, albeit at a slightly reduced Cul3 protein level. This observation could be related to the function of CSN because there is a significant decrease in the total amount of Cullins in CSN mutant cells. Both CSN and Cand1 proteins have been proposed to be involved in the cycle of assembly and disassembly of the CRL complex. This model explains how Cand1 and CSN have paradoxical effects on CRL activity and insists that Cand1-mediated cycling is required for optimal CRL activity. However, the data do not support this cycling model, in which loss of Cand1 enhances the degradation of CiFL as a result of increased activity of CRLs. The double-mutant analyses suggest that regulation of the neddylation pathway is a major mechanism for CiFL degradation. Loss of Cand1 failed to suppress accumulation of CiFL protein in Nedd8AN015 or CSN5null mutant clones. The functions of Nedd8 and CSN with regard to Cullin seem to play a more dominant role in regulating CRLs than that of regulation by Cand1. This could explain why overexpression of Cand1 in CSN5null mutant causes an increase only in the neddylated forms of Cullin, although Cand1 stabilizes unneddylated Cullin (Kim, 2010).
Inhibition of proteasome function by overexpressing a dominant-negative form of a proteasome subunit causes accumulation of unneddylated Cul3. The neddylation defective dAPP-BP1 mutant also exhibits elevated levels of unneddylated Cul3, but repressed proteasomal activity in the dAPP-BP1null mutant fails to causes Cul3 accumulation. These results support the theory that unneddylated Cul3 is degraded by the proteasome, but this degradation effect is inhibited by mutation of the Nedd8 E1-activating enzyme, dAPP-BP1. Accumulation of Cul3 in the dAPP-BP1 mutant is suppressed by loss of Cand1, and decreased Cul3 in the dAPP-BP1, Cand1 double-mutant is again accumulated by reducing proteasome activity. This shows that Cand1 is responsible for the accumulation of unneddylated Cul3 in the dAPP-BP1 mutant as a result of inhibition of proteasome-mediated degradation. Repression of proteasome function in the Cand1 mutant induces accumulation of unneddylated Cul3, showing that neddylated Cul3 is destabilized by the proteasome in the absence of Cand1 (Kim, 2010).
Recent reports indicate that supplementation of substrate and adaptor to Cullin-RING ligases promotes Cullin neddylation and dissociation of the Cullin-Cand1 complex. In agreement with the previous reports, the current data also suggest that the neddylation process might regulate the dissociation of Cul3 from Cand1. If Cand1 is dissociated from Cullin by neddylation, a defect in the neddylation process might promote the interaction of Cand1 with unneddylated Cul3. This could explain why the level of Cul3 was not affected by overexpression of Cand1 in the dAPP-BP1 null mutant, even if Cand1 overexpression increases Cul3 protein levels in the dAPP-BP1null heterozygote background (Kim, 2010).
Although Cand1 affects mostly the Cul3 protein, it also influences the Cul1 protein. Cand1 can bind to Cul1 and the overexpression of Cand1 induces the stabilization of Cul1 as well as Cul3. However, the effect of Cand1 on Cullins seems to differ depending on the type of tissue. Immunoblot analysis of Cand116 extracts from third-instar brain lobes and eye discs showed no distinguishable effect on the ratio of neddylated Cul1, but loss of Cand1 caused a reduction of CiFL protein in the anterior region of the wing disc, where the Cul1-dependent E3 ligase degrades CiFL protein (Kim, 2010).
It is proposed that Cand1 contributes to the fine-tuning of Cul3-mediated E3 ligase activity by acting on the neddylation state as well as on the stability of unneddylated Cul3 and adaptor protein. Binding of Cand1 to unneddylated Cul3 would shift the equilibrium away from the neddylation of Cul3 that is required for substrate degradation and then cause sequestration of unneddylated Cul3 from proteasomal degradation. Moreover, Cand1 could be involved in the suppression of Cul3 adaptor protein, HIB/rdx, to regulate CRL activity. Loss of Cand1 shifts the equilibrium toward the neddylated form of Cul3 and increases the level of Cul3 adaptor HIB/rdx, which leads to enhanced degradation of CiFL, a substrate of CRLs. Neddylation of Cul3 is essential for CRL activity, so the mutation of Cand1 fails to down-regulate accumulation of CiFL in Nedd8 or CSN5 mutants. In the absence of dAPP-BP1, unneddylated Cul3 would tend to bind to Cand1, which protects unneddylated Cul3 from proteasomal degradation and induces accumulation of Cul3 (Kim, 2010).
The mechanisms underlying the Cullin neddylation pathway are closely conserved in Drosophila and in mammals. Consequently, the study of Drosophila Cand1 and Cullin provides a novel insight into the regulation of Cullin based E3 ligases by Cand1 (Kim, 2010).
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