InteractiveFly: GeneBrief

Cullin-3: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Ubiquitin-mediated proteolysis regulates the steady-state abundance of proteins and controls cellular homoeostasis by abrupt elimination of key effector proteins. A multienzyme system targets proteins for destruction through the covalent attachment of a multiubiquitin chain. The specificity and timing of protein ubiquitination is controlled by ubiquitin ligases, such as the Skp1-Cullin-F box protein complex. Cullins are major components of SCF complexes, and have been implicated in degradation of key regulatory molecules including Cyclin E, beta-catenin and Cubitus interruptus. The Drosophila Cullin-3 homologue, Guftagu, has been genetically identified and molecularly characterized. Perturbation of Cullin-3 function has pleiotropic effects during development, including defects in external sensory organ development, pattern formation and cell growth and survival. Loss or overexpression of Cullin-3 causes an increase or decrease, respectively, in external sensory organ formation, implicating Cullin-3 function in regulating the commitment of cells to the neural fate. Cullin-3 function modulates Hedgehog signalling by regulating the stability of full-length Cubitus interruptus (Ci155). Loss of Cullin-3 function in eye discs but not other imaginal discs promotes cell-autonomous accumulation of Ci155. Conversely, overexpression of Cullin-3 results in a cell-autonomous stabilisation of Ci155 in wing, haltere and leg (but not eye), imaginal discs suggesting tissue-specific regulation of Cullin-3 function. The diverse nature of Cullin-3 phenotypes highlights the importance of targeted proteolysis during Drosophila development (Mistry, 2004).

Targeted degradation of short-lived proteins is a universal process that regulates diverse cellular functions. A key example of the importance of targeted protein degradation comes from the observation that the rapid and timely destruction of Cyclin proteins regulates cell cycle progression. Cyclins and other proteins are marked for destruction by the action of a ubiquitin ligase whose specific targeting activity mediates the covalent attachment of a ubiquitin polymer to select residues of the target protein. Four types of ubiquitin ligases have been identified: (1) the N-end rule/Ubr1 ligase, (2) the HECT-domain family, (3) the Cyclosome/Anaphase Promoting Complex and the (4) Skp1-Cullin-F box/Elongin C-Cullin-SOCS box (SCF/ECS) complex (Deshaies, 1999; Hershko, 1998; see Drosophila Slimb). Of these, the SCF/ECS complex was the first identified and is the best understood (Mistry, 2004).

SCF and ECS complexes (Deshaies, 1999 and Tyers, 1999) target distinct groups of proteins for degradation. Cullins are conserved proteins of ~800 residues that comprise the scaffold of both SCF and ECS ubiquitin ligases. Cullins interact with Skp1 or Elongin C homologues through their N terminus and with Hrt1/Roc1/Rbx1, a RING-finger-containing protein through their C-terminus. Skp1-like or Elongin C-like proteins interact with F-box or SOCS-box containing proteins, which target specific proteins for ubiquitination by their respective ubiquitin ligase complex. Recently, BTB domain-containing proteins have been shown to interact with Cullin-3 and subsume the role of the Skp1 and F-box proteins in substrate recognition (Geyer, 2003;; Pintard, 2003; Xu, 2003), suggesting the existence of yet another ubiquitin ligase complex, in addition to the four listed above, with a distinct repertoire of protein targets (Geyer, 2003; Pintard, 2003; Xu, 2003). The modular nature of multisubunit ubiquitin ligases endows different complexes with distinct substrate specificity. Targeted protein ubiquitination plays a critical role in mediating the response of multiple developmental signalling pathways. In Drosophila, SCF complexes have been implicated in ubiquitination of protein targets in three signalling pathways; IκBα in the NF-κB pathway; β-catenin/Armadillo in the Wnt pathway and Ci in the Hedgehog pathway. Signal activation in the Hedgehog (Hh) pathway, for example, leads to the tissue-dependent expression of the Wingless (Wg) and Decapentaplegic (Dpp) morphogens (Mistry, 2004).

The Drosophila Cullin-3 homologue (dCul-3) has been identifed and characterized. dCul-3 plays a broad role to regulate the development of many different adult structures. For example, loss of function and over-expression studies indicate that dCul-3 inhibits sensory organ development during adult development consistent with the idea that dCul-3 modulates Notch pathway activity. In addition, genetic studies confirm and extend the relationship between dCul-3 function and the regulation of Ci155 levels and thus, Hedgehog signalling. These results suggest that dCul-3 function impinges on the activity of many different signalling pathways and developmental events via the targeted destruction or modification of specific proteins (Mistry, 2004).

guftagu was identified in a screen for dominant modifiers of an adult viable wing and notal phenotype caused by GAL4::UAS-mediated misexpression of constitutively activated Gα_s (Gα_s*). A collection of overlapping autosomal deficiencies corresponding to ~70% of the autosomal genome was screened to identify genomic regions capable of dominant modification of the Gα_s* phenotype. This screen uncovered 28 genomic regions likely to contain dominant modifiers of the Gα_s* phenotype. Heterozygosity for each of these 28 regions suppresses the Gα_s* phenotype although the extent of suppression varies between regions. To identify the modifying loci, smaller deletions and mutations in individual loci were screened within each genomic region for the ability to modify the Gα_s* phenotype. Seventeen single loci in 13 genomic regions capable of dominant modification of the Gα_s* phenotype were identified. Single modifying loci in the remaining 15 genomic regions were not identified (Mistry, 2004).

Two of the 28 deficiencies, Df(2L)osp29 (35B1; 35E6), and Df(3L)vin7 (69A1-69A5), suppress the Gα_s* phenotype to near wild-type levels. A systematic search of the complementation groups uncovered by Df(2L)osp29 identified l(2)35Cd at polytene position 35C4 in this region as a suppressor of the Gα_s* phenotype. The gene was designated guftagu (gft), an Urdu word that means ‘private conversation’, because it is believed that the gene product has a private conversation with the activated Gα_s signalling pathway in order to modify the Gα_s* phenotype (Mistry, 2004).

Thus dCul-3 was initially detected as a dominant suppressor of Gα_s signal transduction, indicating the ability of dCul-3 to modulate the activity of at least one signalling pathway. Subsequent studies determined that dCul-3 plays a broad role to regulate the development of many different structures during adult development consistent with the idea that dCul-3 activity modulates the strength of multiple signalling pathways during Drosophila development (Mistry, 2004).

Phosphorylation of Ci triggers its subsequent proteolysis. One mechanism that might couple phosphorylation with proteolysis is the ubiquitin-mediated degradation pathway regulated by ubiquitin ligases such as the SCF complex. The F-box-containing factor Slimb is required for the generation of Ci75, the repressor form of Ci. Using the developing eye disc as a model, Ou (2002) has shown that Ci155 stability is controlled differentially by dCul-1 and dCul-3. Slimb and dCul-1 function anterior to the morphogenetic furrow to target Ci155 for proteolysis, while dCul-3 functions posterior to the furrow to mediate the same event (Mistry, 2004).

The current study supports the link between Cullin/SCF function and Ci155 stability during imaginal disc development. Loss of dCul-3 function in posterior compartment cells of the wing disc immediately adjacent to the AP boundary results in a non-autonomous reduction in Ci155 accumulation in anterior compartment cells that abut dCul-3 mutant cells. Furthermore, overexpression of dCul-3 in the anterior but not posterior compartment of wing, haltere and leg imaginal discs leads to a cell-autonomous increase in Ci155 stability. Thus, dCul-1 and dCul-3 are required in distinct developmental contexts to regulate Ci155 stability and Hh signal transduction (Mistry, 2004).

Together with the results of Ou (2002) , these data support the model that dCul-3 functions autonomously to regulate Ci155 stability in a region-specific manner. In the eye, dCul-3 likely acts in a complex to promote the cleavage of Ci155 into the Ci75 repressor form. dCul-3 could mediate this activity directly, by associating with a specific F-box protein that tethers Ci155 to an SCF complex containing dCul-3. Alternatively, dCul-3 could mediate this effect indirectly, by targeted degradation or modification of a protein involved in the regulation of Ci155 stability. In the wing, dCul-3 overexpression could lead to an autonomous accumulation of Ci155 either by titrating other SCF complex components that promote the limited proteolysis of Ci155 to Ci75 or by targeting a protein for degradation that is normally required to promote limited proteolysis of Ci155. At present these data do not distinguish clearly between these models, although the reciprocal phenotypes observed in dCul-3 mutant clones relative to tissues that overexpress dCul-3 suggest dCul-3 does not act solely in a dominant negative manner (Mistry, 2004).

The non-autonomous effect of dCul-3 loss on Ci155 stability suggests that dCul-3 can modulate Ci155 accumulation through multiple mechanisms. In this context, the simplest model is that dCul-3 function is required for the proper expression or transmission of the Hh signal. The apparent ability of dCul-3 to regulate Ci155 stability through at least two different mechanisms and the diversity of dCul-3 phenotypes, suggest that the composition of dCul-3-containing SCF complexes varies in a region- and stage-specific manner. Given this, a clear understanding of the molecular basis through which dCul-3 regulates Ci155 stability as well as the activity/levels of other proteins will require the identification of the direct targets of dCul-3/SCF complexes through biochemical and molecular genetic means (Mistry, 2004).

In addition to its effects on the Hh and Gα_s pathways, loss of dCul3 function results in embryonic and adult phenotypes reminiscent of defects in EGF and Notch signalling. For example, loss of maternal dCul-3 function produces small embryos with fused dorsal appendages, a phenotype similar to that generated by reduction or loss of function in members of the EGF receptor pathway. Loss of dCul-3 function also causes ectopic sensory organ formation and shaft duplication similar to phenotypes that arise as a result of compromised Notch activity. Likewise, the venation phenotypes dCul3 clones are similar to those that arise due to perturbations in either the Notch or EGF-receptor signalling pathways. These results raise the possibility that dCul-3 function also modulates the activity of the Notch and/or EGF-receptor signalling pathways (Mistry, 2004).

Structure function analyses of Cullins identify three distinct domains: an N-terminal domain that binds Skp- or Elongin C-like proteins; a C-terminal domain that binds Hrt1/Roc1/Rbx1 and a ~60 amino acid domain at the extreme C-terminus of unknown function. The latter domain exhibits the highest degree of sequence identity (~50%) between different Cullins and contains the invariant tyrosine-rich motif, Y-X₂-R-X_6-7-Y/F-X-Y-X-A/S, known as the Cullin motif. However, the functional significance of this domain remains unclear. Although dispensable for Cullin function in yeast, the C-terminal domain is modified post-translationally by the covalent attachment of Nedd8, a small, ubiquitin-like protein. An intact neddylation pathway is required for Cullin-dependent ubiquitination of HIFα, p27 and IκBα in mammalian cells and mutations in Cul-1 and nedd8 both lead to heightened accumulation of Ci155 in Drosophila eye discs (Ou, 2002). Therefore, post-translational modification of the extreme C-terminus of Cullins by Nedd8 might be required for SCF-mediated ubiquitination of target proteins (Mistry, 2004 and references therein).

Genetic studies of dCul-3 provide evidence for the in vivo importance of the C-terminal domain. The gft^HG39 lesion encodes a protein with a truncated C terminal domain that is phenotypically one of the most severe dCul-3 alleles, demonstrating the in vivo relevance of the extreme C-terminal domain. Consistent with this, overexpression of a full-length form of dCul-3 is found to be sufficient to induce phenotypes reciprocal to those observed in dCul-3 mutant clones whereas overexpression of a truncated form of dCul-3 that lacks the C-terminal domain yields no overt phenotypes. These data suggest that the dCul-3 C-terminal domain is necessary for dCul-3 activity during Drosophila development. This is the first clear demonstration of the in vivo importance of the C-terminal domain. Future experiments are required to elucidate the precise molecular mechanisms through which the C-terminal domain enables Cullin protein function (Mistry, 2004).

These results demonstrate that the mechanisms by which multisubunit ubiquitin ligases target specific proteins for degradation are complex. For example, dCul-1 and dCul-3 both promote Ci155 proteolysis -- but they do so in distinct spatial domains (Ou, 2002). In addition, complexes containing dCul-1 and dCul-3 can discriminate between distinct forms of the same protein; SCF complexes containing Cul-1 target the phosphorylated form of Cdk2-bound Cyclin E while SCF complexes containing Cul-3 target free unphosphorylated Cyclin E for degradation (Singer, 1999). The specificity of Cul-1 and Cul-3 to target the same protein in different domains and in different conditions, hints at the precision and complexity of targeted protein degradation. Recent work in C. elegans adds another layer of complexity to this equation, as BTB domain-containing proteins, such as MEL-26, appear to identify new adaptor molecules that link Cul-3 with specific target proteins, such as MEI-1, for ubiquitination (Pintard, 2003; Xu, 2003). These data together with the large number of SCF-like components in higher eukaryotes, underline the enormous combinatorial potential for the formation of distinct multisubunit ubiquitin ligases and their ability to target distinct, but potentially overlapping, sets of proteins for degradation (Mistry, 2004).

Cul3 and insomniac are required for rapid ubiquitination of postsynaptic targets and retrograde homeostatic signaling

At the Drosophila neuromuscular junction, inhibition of postsynaptic glutamate receptors activates retrograde signaling that precisely increases presynaptic neurotransmitter release to restore baseline synaptic strength. However, the nature of the underlying postsynaptic induction process remains enigmatic. In this study a forward genetic screen is described to discover factors in the postsynaptic compartment necessary to generate retrograde homeostatic signaling. This approach identified insomniac (inc), a putative adaptor for the Cullin-3 (Cul3) ubiquitin ligase complex, which together with Cul3 is essential for normal sleep regulation. Interestingly, it was found that Inc and Cul3 rapidly accumulate at postsynaptic compartments following acute receptor inhibition and are required for a local increase in mono-ubiquitination. Finally, it was shown that Peflin, a Ca(2+)-regulated Cul3 co-adaptor, is necessary for homeostatic communication, suggesting a relationship between Ca(2+) signaling and control of Cul3/Inc activity in the postsynaptic compartment. This study suggests that Cul3/Inc-dependent mono-ubiquitination, compartmentalized at postsynaptic densities, gates retrograde signaling and provides an intriguing molecular link between the control of sleep and homeostatic plasticity at synapses (Kikuma, 2019).

By screening >300 genes with putative functions at synapses, this study has identified inc as a key postsynaptic regulator of retrograde homeostatic signaling at the Drosophila NMJ. The data suggest that Inc and Cul3 are recruited to the postsynaptic compartment within minutes of glutamate receptor perturbation, where they promote local mono-ubiquitination. Inc/Cul3 appear to function downstream of or in parallel to CaMKII and upstream of retrograde signaling during PHP. Pef was identified as a putative co-adaptor that may work with Inc/Cul3 to link Ca2+ signaling in the postsynaptic compartment with membrane trafficking and retrograde communication. Altogether, these findings implicate a post translational signaling system involving mono-ubiquitination in the induction of retrograde homeostatic signaling at postsynaptic compartments (Kikuma, 2019).

Although forward genetic screens have been very successful in identifying genes required in the presynaptic neuron for the expression of PHP, these screens have provided less insight into the postsynaptic mechanisms that induce retrograde homeostatic signaling. It seems clear that many genes acting presynaptically are individually required for PHP, with loss of any one completely blocking PHP expression. Indeed, ~25 genes that function in neurons have thus far been implicated in PHP expression. In contrast, forward genetic screens have largely failed to uncover new genes functioning in the postsynaptic muscle during PHP, implying some level of redundancy. The specific postsynaptic induction mechanisms driving retrograde PHP signaling have therefore remained unclear, and are further complicated by cap-dependent translation and metabolic pathways that contribute to sustaining PHP expression over chronic, but not acute, time scales. Therefore, it is perhaps not surprising that despite screening hundreds of mutants, this study found only a single gene, insomniac, to be required for PHP induction. Inc is expressed in the nervous system and can traffic to the presynaptic terminals of motor neuron. In the context of PHP signaling, however, inc was found to be required in the postsynaptic compartment, where it functions downstream of or in parallel to CaMKII. One attractive possibility is that a reduction in CaMKII-dependent phosphorylation of postsynaptic targets enables subsequent ubiquitination by Cul3-Inc complexes, and that this modification ultimately drives retrograde signaling during PHP. Indeed, reciprocal influences of phosphorylation and ubiquitination on shared targets are a common regulatory feature in a variety of signaling systems. The dynamic interplay of phosphorylation and ubiquitination in the postsynaptic compartment may enable a sensitive and tunable mechanism for controlling the timing and calibrating the amplitude of retrograde signaling at the NMJ (Kikuma, 2019).

The substrates targeted by Inc and Cul3 during PHP induction are not known, but the identification of mono-ubiquitination in the postsynaptic compartment during PHP signaling and the putative Cul3 co-adaptor Peflin provides a foundation from which to assess possible candidates and pathways. In mammals, Pef forms a complex with another Ca2+ binding protein, ALG2, to confer Ca2+ regulation to membrane trafficking pathways. Moreover, Pef/ALG2 were recently found to serve as target-specific co-adaptors for Cul3-KLHL12. In particular, SEC31 and other components involved in ER-mediated membrane trafficking pathways were shown to be targeted for mono-ubiquitination, which in turn modulate Collagen secretion. One attractive possibility, therefore, is that Cul3/Inc could respond to changes in Ca2+ in the postsynaptic compartment through regulation by Pef during PHP signaling to control membrane trafficking pathways. Importantly, the subsynaptic reticulum (SSR) is a complex and membraneous network at the Drosophila NMJ, where electrical, Ca2+-dependent, and membrane trafficking pathways in the postsynaptic compartment are integrated (Teodoro, 2013; Nguyen, 2016). Indeed, Multiplexin, a fly homolog of Collagen XV/XVIII and a proposed retrograde signal, is secreted into the synaptic cleft and is required for trans-synaptic retrograde signaling during PHP (Wang, 2014). In addition, another proposed retrograde signal and secreted protein, Semaphorin 2B, was recently shown to function postsynaptically in retrograde PHP signaling (Orr, 2017). However, inc does not appear to be the closest Drosophila ortholog to KLHL12, and it is therefore possible that Pef and Cul3/Inc regulate postsynaptic PHP signaling through a more indirect mechanism (Kikuma, 2019).

While the precise relationships between CaMKII, Inc, Cul3, and Pef are currently unclear, the activity of membrane trafficking pathways could ultimately be targeted for modulation by Ca2+- and Cul3/Inc-dependent signaling during PHP induction. First, a role for postsynaptic membrane trafficking and elaboration during PHP signaling has already been suggested. In addition, extracellular Ca2+ does not appear to be involved in rapid PhTx-dependent PHP induction. It is therefore tempting to speculate that Ca2+ release from the postsynaptic SSR during rapid PHP signaling may influence Cul3/Inc activity through Pef-dependent regulation, as transient changes in ER-derived Ca2+-signaling controls Pef-dependent recruitment of Cul3 (McGourty, 2016). Alternatively, postsynaptic scaffolds and/or glutamate receptors themselves may be targeted by Cul3/Inc at the Drosophila NMJ, given that these proteins are involved in ubiquitin-mediated signaling and remodeling at dendritic spines. Consistent with this idea, there is evidence that signaling complexes composed of neurotransmitter receptors, CaMKII, and membrane-associated guanylate kinases are intimately associated at postsynaptic densities in Drosophila, as they are in the mammalian central nervous system. There has been speculation that these complexes are targets for modulation during PHP signaling. Although these models are not mutually exclusive, further studies will be required to determine the specific substrates and signal transduction mechanisms through which Cul3/Inc and Pef initiate and sustain retrograde homeostatic communication in postsynaptic compartments (Kikuma, 2019).

While it is well established that the ubiquitin proteasome system can sculpt and remodel synaptic architecture, the importance of mono-ubiquitination at synapses is less studied. Ubiquitin-dependent pathways play key roles in synaptic structure, function, and degeneration, and also contribute to activity-dependent dendritic growth. However, the fact that some proteins persist for long periods at synapses suggests that modification of these proteins by ubiquitin likely include non-degredative and reversible mechanisms. Indeed, a recent study revealed a remarkable heterogeneity in the stability of synaptic proteins, with some short lived and rapidly turned over, while others persisting for long time scales, with half lives of months or longer. At the Drosophila NMJ, rapid ubiquitin-dependent proteasomal degradation at presynaptic terminals is necessary for the expression of PHP through modulation of the synaptic vesicle pool (Wentzel, 2018). In contrast, postsynaptic proteasomal degradation does not appear to be involved in rapid PHP signaling, suggesting that ubiquitin-dependent pathways in the postsynaptic compartment contribute to PHP signaling by non-degradative mechanisms. The current data demonstrate that Cul3, Inc, and Pef function in muscle to enable retrograde PHP signaling, and suggest that Cul3/Inc rapidly trigger mono-ubiquitination at postsynaptic densities following glutamate receptor perturbation. Interestingly, synaptic proteins can be ubiquitinated in <15 s following depolarization-induced Ca2+ influx at synapses (Chen, 2003) and changes in intracellular Ca2+ can activate Pef and Cul3 signaling with similar rapidity. Therefore, both poly- and mono-ubiquination may function in combination with other rapid and reversible processes, including phosphorylation at postsynaptic compartments to enable robust and diverse signaling outcomes during the induction of homeostatic plasticity (Kikuma, 2019).

A prominent hypothesis postulates that a major function of sleep is to homeostatically regulate synaptic strength following experience-dependent changes that accrue during wakefulness. Several studies have revealed changes in neuronal firing rates and synapses during sleep/wake behavior, yet few molecular mechanisms that directly associate the electrophysiological process of homeostatic synaptic plasticity and sleep have been identified. The finding that inc is required for the homeostatic control of synaptic strength provides an intriguing link to earlier studies, which implicate inc in the regulation of sleep. It remains to be determined to what extent the role of inc in controlling PHP signaling at the NMJ is related to the impact of inc on sleep and, if so, whether Inc targets the same substrates to regulate these processes. Interestingly, virtually all neuropsychiatric disorders are associated with sleep dysfunction, including those associated with homeostatic plasticity and Fragile X Syndrome, and sleep behavior is also disrupted by mutations in the Drosophila homolog of FMRP, dfmr1. Further investigation of this intriguing network of genes involved in the homeostatic control of sleep and synaptic plasticity may help solve the biological mystery that is sleep and also shed light on the etiology of neuropsychiatric diseases (Kikuma, 2019).

Protein Interactions

Distinct protein degradation mechanisms mediated by Cul1 and Cul3 controlling Ci stability in Drosophila eye development

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 SCF^Slimb 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 (Ci^FL) 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 Ci^FL, 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 Ci^FL stability, it was found that there is mechanistic difference in controlling Ci^FL stability between anterior and posterior cells separated by the MF. Whereas the Cul1-based SCF^Slimb complex controls Ci^FL stability in anterior cells, a Cul3-dependent protein degradation mechanism controls Ci^FL stability in posterior cells. The differences between these two protein degradation mechanisms were further investigated (Ou, 2002).

In anterior cells of developing discs, Ci^FL proteolytic processing requires the activity of the Nedd8-modified, Cul1-based SCF^Slimb complex. This Ci^FL proteolytic processing is inhibited by Smo signaling and promoted by PKA phosphorylation on Ci^FL. The mechanism by which Ci^FL is proteolyzed from Ci^FL to Ci75 is not clear. It is proposed that Nedd8 modifies and activates SCF^Slimb for Ci ubiquitination and then proteolysis, as evidenced by Cul1 modification by Nedd8 and Ci^FL accumulation in Nedd8, Cul1, and slimb mutants. Consistently, proteolysis of Ci^FL 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 Ci^FL 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 Ci^FL 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 Ci^FL 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 SCF^Slimb-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 Ci^FL 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 Ci^FL 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. Ci^FL 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 Ci^FL (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 Ci^FL 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 Nedd8^AN015 and Nedd8^AN024. 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 Nedd8^AN015 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 (Ci^FL) 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 Nedd8^AN015/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 Ci^FL 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, Ci^FL accumulates to a level identical to that in the MF cells that transduce the Hh signaling pathway. Accumulation of Ci^FL also exists in posterior mutant cells that locate proximally but not distally to the MF. Ci^FL 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 Ci^FL accumulation (Ou, 2002).

Elevated Ci^FL 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 Ci^FL in Nedd8 mutant cells is able to respond to Hh signaling (Ou, 2002).

Ci^FL accumulation in Nedd8 cells results from a defect in the machinery controlling Ci^FL protein processing. Ci protein processing is known to depend on the phosphorylation status of Ci^FL by PKA. The level of Ci^FL 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 Ci^FL 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, Ci^FL 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 Ci^FL downregulation by PKA activity requires Nedd8 activity, and the effect of the Nedd8 pathway on Ci^FL processing is unlikely to be mediated through modulation of PKA activity (Ou, 2002).

Ci downregulation in the posterior cells of the eye disc requires Smo signaling and Nedd8 modification activity; Ci^FL degradation is mediated by a Cul3-dependent mechanism

The finding that Ci^FL accumulates in posterior smo³ clones indicates that Smo signaling contributes to the downregulation of Ci^FL 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 Ci^FL activation. Ci^FL 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 Ci^FL is further enhanced, even in clones located distally to the MF, whereas no Ci^FL 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 Ci^FL 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 Ci^FL degradation in the eye disc, the available Drosophila Cul3 mutants were analyzed. Ci^FL 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 Ci^FL accumulation is detected in anterior Cul3 mutant clones, indicating that Cul3 controls Ci^FL 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 Ci^FL 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 SCF^Slimb complex that controls Ci^FL 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 Ci^FL processing, and plays a role in the Hh signaling pathway for Ci activation. The requirement of PKA in Ci^FL degradation in the posterior cells of the eye disc was examined; Ci^FL downregulation is not regulated by PKA activity. Proteolytic processing of Ci^FL 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 Ci^FL to the short form Ci75 in the anterior cells. To sum up, the results suggest that in the posterior cells of the eye disc, Ci^FL is degraded constitutively, and this degradation process is independent of PKA phosphorylation (Ou, 2002).

Targeted disruption of Drosophila Roc1b reveals functional differences in the Roc subunit of Cullin-dependent E3 ubiquitin ligases

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).

Roadkill attenuates Hedgehog responses through degradation of Cubitus interruptus involving Cullin 3; Roadkill substrate-specific adaptors for Cullin3-based ubiquitin E3 ligases

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).

A ubiquitin ligase complex regulates caspase activation during sperm differentiation in Drosophila

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 (Cul3^Testis), 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 Cul3^Testis 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).

Gradients of a ubiquitin E3 ligase inhibitor and a caspase inhibitor determine differentiation or death in spermatids

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).

Drosophila Cand1 regulates Cullin3-dependent E3 ligases by affecting the neddylation of Cullin3 and by controlling the stability of Cullin3 and adaptor protein

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 Ci^FL, 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 Ci^FL 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).

Ci^FL is processed by two different Cullins, Cul1 and Cul3, in the eye disc of Drosophila. In the posterior area of the eye imaginal disc, Ci^FL is degraded by Cul3-mediated E3 activity, where loss of Cand1 affects the stability of Ci^FL. Because it was observed that mutation of Cand1 decreases the level of Ci^FL, 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 Ci^FL as a result of increased activity of CRLs. The double-mutant analyses suggest that regulation of the neddylation pathway is a major mechanism for Ci^FL degradation. Loss of Cand1 failed to suppress accumulation of Ci^FL protein in Nedd8^AN015 or CSN5^null 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 CSN5^null 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-BP1^null 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-BP1^null 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 Ci^FL protein in the anterior region of the wing disc, where the Cul1-dependent E3 ligase degrades Ci^FL 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 Ci^FL, a substrate of CRLs. Neddylation of Cul3 is essential for CRL activity, so the mutation of Cand1 fails to down-regulate accumulation of Ci^FL 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).

Cullin-3 controls Timeless oscillations in the Drosophila circadian clock

Eukaryotic circadian clocks rely on transcriptional feedback loops. In Drosophila, the Period and Timeless proteins accumulate during the night, inhibit the activity of the Clock (Clk)/Cycle (Cyc) transcriptional complex, and are degraded in the early morning. The control of Per and Tim oscillations largely depends on post-translational mechanisms. They involve both light-dependent and light-independent pathways that rely on the phosphorylation, ubiquitination, and proteasomal degradation of the clock proteins. Slmb, which is part of a CULLIN-1-based E3 ubiquitin ligase complex, is required for the circadian degradation of phosphorylated Per. This study shows that Cullin-3 (Cul-3) is required for the circadian control of Per and Tim oscillations. Expression of either Cul-3 RNAi or dominant negative forms of Cul-3 in the clock neurons alters locomotor behavior and dampens Per and Tim oscillations in light-dark cycles. In constant conditions, Cul-3 deregulation induces behavioral arrhythmicity and rapidly abolishes Tim cycling, with slower effects on Per. Cul-3 affects Tim accumulation more strongly in the absence of Per and forms protein complexes with hypo-phosphorylated Tim. In contrast, Slmb affects Tim more strongly in the presence of Per and preferentially associates with phosphorylated Tim. Cul-3 and Slmb show additive effects on Tim and Per, suggesting different roles for the two ubiquitination complexes on Per and Tim cycling. This work thus shows that Cul-3 is a new component of the Drosophila clock, which plays an important role in the control of Tim oscillations (Grima, 2012).

Cul3 and the BTB adaptor insomniac are key regulators of sleep homeostasis and a dopamine arousal pathway in Drosophila

Sleep is homeostatically regulated, such that sleep drive reflects the duration of prior wakefulness. However, despite the discovery of genes important for sleep, a coherent molecular model for sleep homeostasis has yet to emerge. To better understand the function and regulation of sleep, a reverse-genetics approach was employed in Drosophila. An insertion in the BTB domain protein CG32810/insomniac (inc) exhibited one of the strongest baseline sleep phenotypes thus far observed, a ~10 h sleep reduction. Importantly, this is coupled to a reduced homeostatic response to sleep deprivation, consistent with a disrupted sleep homeostat. Knockdown of the INC-interacting protein, the E3 ubiquitin ligase Cul3, results in reduced sleep duration, consolidation, and homeostasis, suggesting an important role for protein turnover in mediating INC effects. Interestingly, inc and Cul3 expression in post-mitotic neurons during development contributes to their adult sleep functions. Similar to flies with increased dopaminergic signaling, loss of inc and Cul3 result in hyper-arousability to a mechanical stimulus in adult flies. Furthermore, the inc sleep duration phenotype can be rescued by pharmacological inhibition of tyrosine hydroxylase, the rate-limiting enzyme for dopamine biosynthesis. Taken together, these results establish inc and Cul3 as important new players in setting the sleep homeostat and a dopaminergic arousal pathway in Drosophila (Pfeiffenberger, 2012).

Using genetic backcrossing to an isogenic (iso31) strain, the striking observation was made that the vast majority of the mutants (39/45, nearly 90%) identified in a primary genetic screen did not have a significant phenotype after backcrossing, indicating a remarkably pervasive role for genetic background in mediating sleep phenotypes in a variety of mutant strains. There are two main possibilities for how genetic background influences sleep phenotypes: (1) the tested allele indeed affects sleep; however, there are suppressors of this phenotype present in the iso31 background but absent from the original background. (2) The sleep phenotype is not due to the transposon insertion but instead is caused by one or more flanking mutations present in the original mutant background but absent from the iso31 background. Isolated examples of (1) have been observed in the case of Sh and mutants of the Sh regulatory subunit Hyperkinetic as well as Crc and Sema-5c effects on olfactory, startle, and sleep behavior and of (2) in the discovery of DAT^fmn mutants in the background of a timeless mutant strain. These are consistent with observations in C. elegans indicating the limitations of backcrossing for removing flanking mutations and in Drosophila indicating the widespread presence of background mutations that can suppress mutant-induced behavioral phenotypes. The experience with RhoGDI^EY02738 suggests that scenario 2 may be more common than previously thought. Nonetheless, the sheer number of examples observed in this study indicates that the presence of genetic variation at sleep regulatory loci among laboratory stocks is both prevalent and perhaps even sufficiently important to mask or induce significant sleep phenotypes. Moreover, in the case of Sh a single outcross was sufficient to unmask the short sleep phenotype. Indeed, it was assumed that if mutant alleles were outcrossed to a deficiency strain this would effectively remove the influence of accumulated recessive mutations that flank the allele. However, backcrossing data indicates that this strategy did not remove those concerns, suggesting that background variants may exert dominant effects. Practically, the experience suggests that outcrossing to deletion stocks alone may not be sufficient to verify the function of a genetic locus in sleep. Overall, this observation has important implications for the role of genetic modifiers in sleep, the conduct and design of sleep genetic screens, and for the interpretation of sleep and other behavioral mutant phenotypes in general. While backcrossing can remove flanking genetic variants that may contribute to an observed phenotype, alone it is not sufficient to definitively establish genotype-phenotype causation (Pfeiffenberger, 2012).

Despite the large modulatory effect of genetic background, it was possible to observe persistent phenotypes with inc, which showed the most robust and reproducible sleep phenotypes, in particular demonstrating an important role in the homeostatic regulation of sleep. Several independent lines of evidence support the role of inc in sleep homeostasis. First, two inc alleles (inc^f00285 and inc^mw) were backcrossed for 5 generations into an isogenic background, and they retained their short sleep and suppressed sleep homeostasis phenotypes, each among the strongest observed, as compared to isogenic control lines. Second, inc^f00285 was rescued in 2 distinct ways: (1) with genomic duplications encompassing the gene but not those that do not include the gene, and (2) using the GAL4/UAS system, the latter rescuing both baseline and homeostatic phenotypes. Third, failure to complement was demonstrated with a deletion removing the inc genomic locus, or inc transheterozygotes. Fourth, it was demonstrated that two independent RNAi lines that target two different regions of inc phenocopy the inc mutant phenotype (Pfeiffenberger, 2012).

In addition, evidence is provided that the INC-interacting protein, the E3 ubiquitin ligase CUL3, functions to regulate sleep levels suggesting that inc links protein turnover to sleep homeostasis. Two independent inserts of a Cul3-RNAi line that effectively suppress Cul3 mRNA levels resulted in reduced sleep, and induction of a wild-type Cul3 transgene could rescue these phenotypes. The CUL3/INC interaction was varified in S2 cells, and synthetic genetic interactions between Cul3 and inc were observed using RNAi, consistent with the model that they operate together to affect sleep (Pfeiffenberger, 2012).

A core concept in understanding sleep behavior is its homeostatic regulation, i.e., the observation that the drive to sleep reflects the duration of prior wakefulness. Sleep homeostasis typically is measured by enforcing wakefulness/depriving sleep for a defined period and assaying the increase in subsequent rebound sleep. Importantly, this study demonstrated that both inc and Cul3 have robust effects on sleep homeostasis where reduced inc or Cul3 was accompanied by suppressed or absent sleep rebound under the conditions used. These results suggest that inc and Cul3, and by extension, protein degradation, are important for the accumulation of sleep need during wake and/or dissipation of sleep need after deprivation (Pfeiffenberger, 2012).

For the large majority of sleep mutants that have been described, assessment of developmental and adult contributions has not formally been addressed, raising questions regarding their precise function in sleep. This study has provided evidence that inc induction or Cul3-RNAi knockdown during development, but not exclusively during adulthood, could rescue (in the case of induction) or phenocopy (in the case of knockdown) their respective mutant/RNAi phenotypes. The Cul3 results are consistent with an established role for Cul3 in dendritic and axonal arborization, in which dendritic and axonal arborization are reduced in Cul3 mutants. The data also revealed a stochastic branching defect in MB neurons in 26% of inc mutants, in which they lack a single α- or β-lobe. Based on the incomplete penetrance of this morphological defect, it cannot explain the sleep behavior phenotype; however, it may be reflective of other morphological phenotypes that are causative for behavior. Alternatively, the necessity for developmental expression of Cul3 and inc may be for the appropriate processing, maturation and/or localization of these proteins in the adult. The apparent long half-life/persistence of this protein after induction only during development is consistent with the possibility that developmentally expressed transcription is important for adult protein expression and function. Regardless, it will be of interest to examine the relative adult and developmental requirements of other sleep mutants (Pfeiffenberger, 2012).

This study found that the reduced sleep phenotype depends largely on a single neurotransmitter, dopamine, establishing a transmitter basis to inc/Cul3 function. Dopaminergic signaling is a key regulator of sleep/wake behavior. In humans, sleep deprivation has been associated with increased brain levels of dopamine. Treatment of Parkinson's disease with L-DOPA can alleviate daytime sleepiness, or in the extreme result in insomnia. In Drosophila, genetic loss or pharmacological inhibition of tyrosine hydroxylase increases sleep. Furthermore, flies that lack a functional copy of DopR exhibit increased sleep and general arousal defects, including reduced arousing effects of caffeine. Conversely, in DAT^fmn flies, or flies fed dopamine-enhancing methamphetamine, sleep levels are severely reduced. Dopamine arousal effects are modulated by light. Moreover, sleep deprivation induced reductions in learning can be suppressed by enhancing dopaminergic signaling. Other than dopamine receptors and DAT, members of the dopaminergic arousal pathway remain largely unknown (Pfeiffenberger, 2012).

This study shows that inc and Cul3 function in the dopaminergic arousal pathway. First, inc mutants, Cul3-RNAi, and DAT^fmn all showed robust sleep duration and consolidation phenotypes. Second, all three groups were hyper-arousable to mechanical stimuli. Third, disruption of inc, Cul3, and DAT all exhibited suppressed or absent homeostatic responses to sleep deprivation. Fourth, the short-sleep phenotypes of inc and DAT^fmn were non-additive in double mutants. Fifth, while wild-type flies exhibited reduced sleep when fed the dopamine precursor L-DOPA, inc mutants were resistant to these effects, but not the arousing effects of the Rdl antagonist CBZ. Finally, the sleep duration phenotypes in flies with disrupted inc, Cul3, and DAT could be suppressed by pharmacologically inhibiting dopamine synthesis with 3IY or AMPT, linking short sleep to excess dopamine function. Importantly, it was demonstrated that inhibition of dopamine synthesis via tyrosine hydroxylase inhibition does not affect L-DOPA-induced sleep reductions. It was also observed that 3IY could restore sleep homeostasis to Cul3-RNAi. Similar 3IY effects on homeostasis were only observed in one of the two inc alleles. Nonetheless, these studies do further link dopamine signaling to sleep homeostasis. inc and Cul3 are the first genes that are not known dopamine receptors reported to function in the dopaminergic arousal pathway, further reinforcing the pivotal role of dopamine in sleep homeostasis (Pfeiffenberger, 2012).

The data suggests Cul3/inc function to regulate dopaminergic signaling downstream of dopamine. inc phenotypes did not map to dopaminergic neurons nor was it possible to identify consistent changes in global dopamine levels among Cul3-RNAi and inc mutant. Thus, Cul3/inc may be involved in active turnover of dopamine receptors or their effectors in neurons defined by Cha-GAL4 and 30Y-GAL4. Double mutants of inc and a major dopamine receptor involved in arousal in Drosophila, DopR, were examined, and no suppression was observed of inc baseline phenotypes; moreover, it was found that DopR mutant flies were responsive to 3IY consumption (i.e. exhibit increased sleep), suggesting that additional dopamine receptors function in Cul3/inc-based dopamine arousal. Drosophila has 2 other dopamine receptors and partial suppression was observed of inc with DopR and DopR2 RNAi, suggesting that multiple dopamine receptors may contribute to these effects. Alternatively, Cul3/inc may be important for protein turnover of other homeostatically regulated components. For example, extensive and dose-dependent changes in synaptic protein expression throughout the brain with sleep deprivation and recovery may depend on Cul3/inc-dependent turnover of these proteins during sleep (Pfeiffenberger, 2012).

Interestingly, Cul3 has also been linked to sleep behavior via a candidate gene for Restless Leg Syndrome (RLS) and BTB gene, BTBD9 (Freeman, 2012). Unlike the current studies, disruption of the Drosophila BTBD9 is not associated with reduced sleep, a reduced level of waking activity, nor elevated dopaminergic signaling. In addition, the phenotypes map in part to dopaminergic neurons in the case of BTBD9 rather than cholinergic neurons for inc. Thus, Cul3/inc likely represents a distinct pathway regulating sleep. Nonetheless, these studies further highlight the importance of Cul3/BTB adaptor pathways in sleep regulation in both Drosophila and humans. Future work will be required to identify the dopamine and sleep-relevant ubiquitination target(s) of inc and Cul3 (Pfeiffenberger, 2012).

REGULATION

Protein Interactions

Distinct protein degradation mechanisms mediated by Cul1 and Cul3 controlling Ci stability in Drosophila eye development

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 SCF^Slimb 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 (Ci^FL) 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 Ci^FL, 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 Ci^FL stability, it was found that there is mechanistic difference in controlling Ci^FL stability between anterior and posterior cells separated by the MF. Whereas the Cul1-based SCF^Slimb complex controls Ci^FL stability in anterior cells, a Cul3-dependent protein degradation mechanism controls Ci^FL stability in posterior cells. The differences between these two protein degradation mechanisms were further investigated (Ou, 2002).

In anterior cells of developing discs, Ci^FL proteolytic processing requires the activity of the Nedd8-modified, Cul1-based SCF^Slimb complex. This Ci^FL proteolytic processing is inhibited by Smo signaling and promoted by PKA phosphorylation on Ci^FL. The mechanism by which Ci^FL is proteolyzed from Ci^FL to Ci75 is not clear. It is proposed that Nedd8 modifies and activates SCF^Slimb for Ci ubiquitination and then proteolysis, as evidenced by Cul1 modification by Nedd8 and Ci^FL accumulation in Nedd8, Cul1, and slimb mutants. Consistently, proteolysis of Ci^FL 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 Ci^FL 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 Ci^FL 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 Ci^FL 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 SCF^Slimb-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 Ci^FL 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 Ci^FL 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. Ci^FL 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 Ci^FL (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 Ci^FL 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 Nedd8^AN015 and Nedd8^AN024. 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 Nedd8^AN015 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 (Ci^FL) 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 Nedd8^AN015/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 Ci^FL 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, Ci^FL accumulates to a level identical to that in the MF cells that transduce the Hh signaling pathway. Accumulation of Ci^FL also exists in posterior mutant cells that locate proximally but not distally to the MF. Ci^FL 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 Ci^FL accumulation (Ou, 2002).

Elevated Ci^FL 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 Ci^FL in Nedd8 mutant cells is able to respond to Hh signaling (Ou, 2002).

Ci^FL accumulation in Nedd8 cells results from a defect in the machinery controlling Ci^FL protein processing. Ci protein processing is known to depend on the phosphorylation status of Ci^FL by PKA. The level of Ci^FL 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 Ci^FL 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, Ci^FL 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 Ci^FL downregulation by PKA activity requires Nedd8 activity, and the effect of the Nedd8 pathway on Ci^FL processing is unlikely to be mediated through modulation of PKA activity (Ou, 2002).

Ci downregulation in the posterior cells of the eye disc requires Smo signaling and Nedd8 modification activity; Ci^FL degradation is mediated by a Cul3-dependent mechanism

The finding that Ci^FL accumulates in posterior smo³ clones indicates that Smo signaling contributes to the downregulation of Ci^FL 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 Ci^FL activation. Ci^FL 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 Ci^FL is further enhanced, even in clones located distally to the MF, whereas no Ci^FL 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 Ci^FL 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 Ci^FL degradation in the eye disc, the available Drosophila Cul3 mutants were analyzed. Ci^FL 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 Ci^FL accumulation is detected in anterior Cul3 mutant clones, indicating that Cul3 controls Ci^FL 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 Ci^FL 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 SCF^Slimb complex that controls Ci^FL 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 Ci^FL processing, and plays a role in the Hh signaling pathway for Ci activation. The requirement of PKA in Ci^FL degradation in the posterior cells of the eye disc was examined; Ci^FL downregulation is not regulated by PKA activity. Proteolytic processing of Ci^FL 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 Ci^FL to the short form Ci75 in the anterior cells. To sum up, the results suggest that in the posterior cells of the eye disc, Ci^FL is degraded constitutively, and this degradation process is independent of PKA phosphorylation (Ou, 2002).

Targeted disruption of Drosophila Roc1b reveals functional differences in the Roc subunit of Cullin-dependent E3 ubiquitin ligases

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).

Roadkill attenuates Hedgehog responses through degradation of Cubitus interruptus involving Cullin 3; Roadkill substrate-specific adaptors for Cullin3-based ubiquitin E3 ligases

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).

A ubiquitin ligase complex regulates caspase activation during sperm differentiation in Drosophila

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 (Cul3^Testis), 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 Cul3^Testis 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).

Gradients of a ubiquitin E3 ligase inhibitor and a caspase inhibitor determine differentiation or death in spermatids

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).

Drosophila Cand1 regulates Cullin3-dependent E3 ligases by affecting the neddylation of Cullin3 and by controlling the stability of Cullin3 and adaptor protein

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 Ci^FL, 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 Ci^FL 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).

Ci^FL is processed by two different Cullins, Cul1 and Cul3, in the eye disc of Drosophila. In the posterior area of the eye imaginal disc, Ci^FL is degraded by Cul3-mediated E3 activity, where loss of Cand1 affects the stability of Ci^FL. Because it was observed that mutation of Cand1 decreases the level of Ci^FL, 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 Ci^FL as a result of increased activity of CRLs. The double-mutant analyses suggest that regulation of the neddylation pathway is a major mechanism for Ci^FL degradation. Loss of Cand1 failed to suppress accumulation of Ci^FL protein in Nedd8^AN015 or CSN5^null 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 CSN5^null 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-BP1^null 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-BP1^null 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 Ci^FL protein in the anterior region of the wing disc, where the Cul1-dependent E3 ligase degrades Ci^FL 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 Ci^FL, a substrate of CRLs. Neddylation of Cul3 is essential for CRL activity, so the mutation of Cand1 fails to down-regulate accumulation of Ci^FL 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).

Cullin-3 controls Timeless oscillations in the Drosophila circadian clock

Eukaryotic circadian clocks rely on transcriptional feedback loops. In Drosophila, the Period and Timeless proteins accumulate during the night, inhibit the activity of the Clock (Clk)/Cycle (Cyc) transcriptional complex, and are degraded in the early morning. The control of Per and Tim oscillations largely depends on post-translational mechanisms. They involve both light-dependent and light-independent pathways that rely on the phosphorylation, ubiquitination, and proteasomal degradation of the clock proteins. Slmb, which is part of a CULLIN-1-based E3 ubiquitin ligase complex, is required for the circadian degradation of phosphorylated Per. This study shows that Cullin-3 (Cul-3) is required for the circadian control of Per and Tim oscillations. Expression of either Cul-3 RNAi or dominant negative forms of Cul-3 in the clock neurons alters locomotor behavior and dampens Per and Tim oscillations in light-dark cycles. In constant conditions, Cul-3 deregulation induces behavioral arrhythmicity and rapidly abolishes Tim cycling, with slower effects on Per. Cul-3 affects Tim accumulation more strongly in the absence of Per and forms protein complexes with hypo-phosphorylated Tim. In contrast, Slmb affects Tim more strongly in the presence of Per and preferentially associates with phosphorylated Tim. Cul-3 and Slmb show additive effects on Tim and Per, suggesting different roles for the two ubiquitination complexes on Per and Tim cycling. This work thus shows that Cul-3 is a new component of the Drosophila clock, which plays an important role in the control of Tim oscillations (Grima, 2012).

Cul3 and the BTB adaptor insomniac are key regulators of sleep homeostasis and a dopamine arousal pathway in Drosophila

Sleep is homeostatically regulated, such that sleep drive reflects the duration of prior wakefulness. However, despite the discovery of genes important for sleep, a coherent molecular model for sleep homeostasis has yet to emerge. To better understand the function and regulation of sleep, a reverse-genetics approach was employed in Drosophila. An insertion in the BTB domain protein CG32810/insomniac (inc) exhibited one of the strongest baseline sleep phenotypes thus far observed, a ~10 h sleep reduction. Importantly, this is coupled to a reduced homeostatic response to sleep deprivation, consistent with a disrupted sleep homeostat. Knockdown of the INC-interacting protein, the E3 ubiquitin ligase Cul3, results in reduced sleep duration, consolidation, and homeostasis, suggesting an important role for protein turnover in mediating INC effects. Interestingly, inc and Cul3 expression in post-mitotic neurons during development contributes to their adult sleep functions. Similar to flies with increased dopaminergic signaling, loss of inc and Cul3 result in hyper-arousability to a mechanical stimulus in adult flies. Furthermore, the inc sleep duration phenotype can be rescued by pharmacological inhibition of tyrosine hydroxylase, the rate-limiting enzyme for dopamine biosynthesis. Taken together, these results establish inc and Cul3 as important new players in setting the sleep homeostat and a dopaminergic arousal pathway in Drosophila (Pfeiffenberger, 2012).

Using genetic backcrossing to an isogenic (iso31) strain, the striking observation was made that the vast majority of the mutants (39/45, nearly 90%) identified in a primary genetic screen did not have a significant phenotype after backcrossing, indicating a remarkably pervasive role for genetic background in mediating sleep phenotypes in a variety of mutant strains. There are two main possibilities for how genetic background influences sleep phenotypes: (1) the tested allele indeed affects sleep; however, there are suppressors of this phenotype present in the iso31 background but absent from the original background. (2) The sleep phenotype is not due to the transposon insertion but instead is caused by one or more flanking mutations present in the original mutant background but absent from the iso31 background. Isolated examples of (1) have been observed in the case of Sh and mutants of the Sh regulatory subunit Hyperkinetic as well as Crc and Sema-5c effects on olfactory, startle, and sleep behavior and of (2) in the discovery of DAT^fmn mutants in the background of a timeless mutant strain. These are consistent with observations in C. elegans indicating the limitations of backcrossing for removing flanking mutations and in Drosophila indicating the widespread presence of background mutations that can suppress mutant-induced behavioral phenotypes. The experience with RhoGDI^EY02738 suggests that scenario 2 may be more common than previously thought. Nonetheless, the sheer number of examples observed in this study indicates that the presence of genetic variation at sleep regulatory loci among laboratory stocks is both prevalent and perhaps even sufficiently important to mask or induce significant sleep phenotypes. Moreover, in the case of Sh a single outcross was sufficient to unmask the short sleep phenotype. Indeed, it was assumed that if mutant alleles were outcrossed to a deficiency strain this would effectively remove the influence of accumulated recessive mutations that flank the allele. However, backcrossing data indicates that this strategy did not remove those concerns, suggesting that background variants may exert dominant effects. Practically, the experience suggests that outcrossing to deletion stocks alone may not be sufficient to verify the function of a genetic locus in sleep. Overall, this observation has important implications for the role of genetic modifiers in sleep, the conduct and design of sleep genetic screens, and for the interpretation of sleep and other behavioral mutant phenotypes in general. While backcrossing can remove flanking genetic variants that may contribute to an observed phenotype, alone it is not sufficient to definitively establish genotype-phenotype causation (Pfeiffenberger, 2012).

Despite the large modulatory effect of genetic background, it was possible to observe persistent phenotypes with inc, which showed the most robust and reproducible sleep phenotypes, in particular demonstrating an important role in the homeostatic regulation of sleep. Several independent lines of evidence support the role of inc in sleep homeostasis. First, two inc alleles (inc^f00285 and inc^mw) were backcrossed for 5 generations into an isogenic background, and they retained their short sleep and suppressed sleep homeostasis phenotypes, each among the strongest observed, as compared to isogenic control lines. Second, inc^f00285 was rescued in 2 distinct ways: (1) with genomic duplications encompassing the gene but not those that do not include the gene, and (2) using the GAL4/UAS system, the latter rescuing both baseline and homeostatic phenotypes. Third, failure to complement was demonstrated with a deletion removing the inc genomic locus, or inc transheterozygotes. Fourth, it was demonstrated that two independent RNAi lines that target two different regions of inc phenocopy the inc mutant phenotype (Pfeiffenberger, 2012).

In addition, evidence is provided that the INC-interacting protein, the E3 ubiquitin ligase CUL3, functions to regulate sleep levels suggesting that inc links protein turnover to sleep homeostasis. Two independent inserts of a Cul3-RNAi line that effectively suppress Cul3 mRNA levels resulted in reduced sleep, and induction of a wild-type Cul3 transgene could rescue these phenotypes. The CUL3/INC interaction was varified in S2 cells, and synthetic genetic interactions between Cul3 and inc were observed using RNAi, consistent with the model that they operate together to affect sleep (Pfeiffenberger, 2012).

A core concept in understanding sleep behavior is its homeostatic regulation, i.e., the observation that the drive to sleep reflects the duration of prior wakefulness. Sleep homeostasis typically is measured by enforcing wakefulness/depriving sleep for a defined period and assaying the increase in subsequent rebound sleep. Importantly, this study demonstrated that both inc and Cul3 have robust effects on sleep homeostasis where reduced inc or Cul3 was accompanied by suppressed or absent sleep rebound under the conditions used. These results suggest that inc and Cul3, and by extension, protein degradation, are important for the accumulation of sleep need during wake and/or dissipation of sleep need after deprivation (Pfeiffenberger, 2012).

For the large majority of sleep mutants that have been described, assessment of developmental and adult contributions has not formally been addressed, raising questions regarding their precise function in sleep. This study has provided evidence that inc induction or Cul3-RNAi knockdown during development, but not exclusively during adulthood, could rescue (in the case of induction) or phenocopy (in the case of knockdown) their respective mutant/RNAi phenotypes. The Cul3 results are consistent with an established role for Cul3 in dendritic and axonal arborization, in which dendritic and axonal arborization are reduced in Cul3 mutants. The data also revealed a stochastic branching defect in MB neurons in 26% of inc mutants, in which they lack a single α- or β-lobe. Based on the incomplete penetrance of this morphological defect, it cannot explain the sleep behavior phenotype; however, it may be reflective of other morphological phenotypes that are causative for behavior. Alternatively, the necessity for developmental expression of Cul3 and inc may be for the appropriate processing, maturation and/or localization of these proteins in the adult. The apparent long half-life/persistence of this protein after induction only during development is consistent with the possibility that developmentally expressed transcription is important for adult protein expression and function. Regardless, it will be of interest to examine the relative adult and developmental requirements of other sleep mutants (Pfeiffenberger, 2012).

This study found that the reduced sleep phenotype depends largely on a single neurotransmitter, dopamine, establishing a transmitter basis to inc/Cul3 function. Dopaminergic signaling is a key regulator of sleep/wake behavior. In humans, sleep deprivation has been associated with increased brain levels of dopamine. Treatment of Parkinson's disease with L-DOPA can alleviate daytime sleepiness, or in the extreme result in insomnia. In Drosophila, genetic loss or pharmacological inhibition of tyrosine hydroxylase increases sleep. Furthermore, flies that lack a functional copy of DopR exhibit increased sleep and general arousal defects, including reduced arousing effects of caffeine. Conversely, in DAT^fmn flies, or flies fed dopamine-enhancing methamphetamine, sleep levels are severely reduced. Dopamine arousal effects are modulated by light. Moreover, sleep deprivation induced reductions in learning can be suppressed by enhancing dopaminergic signaling. Other than dopamine receptors and DAT, members of the dopaminergic arousal pathway remain largely unknown (Pfeiffenberger, 2012).

This study shows that inc and Cul3 function in the dopaminergic arousal pathway. First, inc mutants, Cul3-RNAi, and DAT^fmn all showed robust sleep duration and consolidation phenotypes. Second, all three groups were hyper-arousable to mechanical stimuli. Third, disruption of inc, Cul3, and DAT all exhibited suppressed or absent homeostatic responses to sleep deprivation. Fourth, the short-sleep phenotypes of inc and DAT^fmn were non-additive in double mutants. Fifth, while wild-type flies exhibited reduced sleep when fed the dopamine precursor L-DOPA, inc mutants were resistant to these effects, but not the arousing effects of the Rdl antagonist CBZ. Finally, the sleep duration phenotypes in flies with disrupted inc, Cul3, and DAT could be suppressed by pharmacologically inhibiting dopamine synthesis with 3IY or AMPT, linking short sleep to excess dopamine function. Importantly, it was demonstrated that inhibition of dopamine synthesis via tyrosine hydroxylase inhibition does not affect L-DOPA-induced sleep reductions. It was also observed that 3IY could restore sleep homeostasis to Cul3-RNAi. Similar 3IY effects on homeostasis were only observed in one of the two inc alleles. Nonetheless, these studies do further link dopamine signaling to sleep homeostasis. inc and Cul3 are the first genes that are not known dopamine receptors reported to function in the dopaminergic arousal pathway, further reinforcing the pivotal role of dopamine in sleep homeostasis (Pfeiffenberger, 2012).

The data suggests Cul3/inc function to regulate dopaminergic signaling downstream of dopamine. inc phenotypes did not map to dopaminergic neurons nor was it possible to identify consistent changes in global dopamine levels among Cul3-RNAi and inc mutant. Thus, Cul3/inc may be involved in active turnover of dopamine receptors or their effectors in neurons defined by Cha-GAL4 and 30Y-GAL4. Double mutants of inc and a major dopamine receptor involved in arousal in Drosophila, DopR, were examined, and no suppression was observed of inc baseline phenotypes; moreover, it was found that DopR mutant flies were responsive to 3IY consumption (i.e. exhibit increased sleep), suggesting that additional dopamine receptors function in Cul3/inc-based dopamine arousal. Drosophila has 2 other dopamine receptors and partial suppression was observed of inc with DopR and DopR2 RNAi, suggesting that multiple dopamine receptors may contribute to these effects. Alternatively, Cul3/inc may be important for protein turnover of other homeostatically regulated components. For example, extensive and dose-dependent changes in synaptic protein expression throughout the brain with sleep deprivation and recovery may depend on Cul3/inc-dependent turnover of these proteins during sleep (Pfeiffenberger, 2012).

Interestingly, Cul3 has also been linked to sleep behavior via a candidate gene for Restless Leg Syndrome (RLS) and BTB gene, BTBD9 (Freeman, 2012). Unlike the current studies, disruption of the Drosophila BTBD9 is not associated with reduced sleep, a reduced level of waking activity, nor elevated dopaminergic signaling. In addition, the phenotypes map in part to dopaminergic neurons in the case of BTBD9 rather than cholinergic neurons for inc. Thus, Cul3/inc likely represents a distinct pathway regulating sleep. Nonetheless, these studies further highlight the importance of Cul3/BTB adaptor pathways in sleep regulation in both Drosophila and humans. Future work will be required to identify the dopamine and sleep-relevant ubiquitination target(s) of inc and Cul3 (Pfeiffenberger, 2012).

DEVELOPMENTAL BIOLOGY

Embryonic

RNA in situ hybridization was used to examine the spatiotemporal expression pattern of dCul-3 transcripts in Drosophila embryos and imaginal discs. dCul-3 mRNA is expressed ubiquitously throughout all stages of embryonic development and in imaginal discs. Notably, high levels of dCul-3 mRNA are observed in 0-1 h old embryos indicating significant maternal deposit of dCul-3 mRNA into oocytes. Consistent with RNA in situ hybridization results, dCul-3 protein is detected in the cytoplasm of all cells in embryos and imaginal discs (Mistry, 2004).

Sequence analysis of the two longest cDNAs has identified a short 80 nucleotide cryptic exon flanked by non-consensus splice sites. Retention of the putative exon and in-frame stop codons would truncate dCul-3 prior to the conserved C-terminal domain whereas utilisation of the non-consensus splice sites would produce full-length dCul-3. Although RT-PCR experiments suggested the possibility of multiple dCul-3 protein isoforms, Western analyses on total protein from wild-type (Oregon-R) embryos using antibodies specific to N- and C-terminus of dCul-3 identified a single 80.9 kDa band that appears to correspond to the full-length form of dCul-3. These results indicate that dCul-3 exists primarily, if not exclusively, as a single protein isoform in Drosophila (Mistry, 2004).

Regulation of Hh signal transduction as Drosophila eye differentiation progresses

Differentiation of the Drosophila retina occurs as a morphogenetic furrow sweeps anteriorly across the eye imaginal disc, driven by Hedgehog secretion from photoreceptor precursors differentiating behind the furrow. A BTB protein, Roadkill, is expressed posterior to the furrow and targets the Hedgehog signal transduction component Cubitus interruptus for degradation by Cullin-3 and the proteosome. Clonal analysis and conditional mutant studies establish that roadkill transcription is activated by the EGF receptor and Ras pathway in most differentiating retinal cells, and by both EGF receptor/Ras and by Hedgehog signaling in cells that remain unspecified. These findings outline a circuit by which Hedgehog signal transduction is modified as Hedgehog signaling initiates retinal differentiation. A model is presented for regulation of the Cullin-3 and Cullin-1 pathways that modifies Hedgehog signaling as the morphogenetic furrow moves and the responses of retinal cells change (Baker, 2009).

As the morphogenetic furrow crosses the eye disc, Ci155 accumulates most highly just anterior to the morphogenetic furrow, even though Hh is secreted posterior to the morphogenetic furrow. The sharp reduction in Ci155 as the furrow passes is associated with a switch from Cul1-dependent processing to Cul3-dependent degradation (Ou, 2002). The posterior eye expresses rdx, encoding a BTB protein that couples Ci 155 to the Cul3 pathway (Kent, 2006; Zhang, 2006). This study identified the signals that induce rdx and that process Ci155 in the posterior eye (Baker, 2009).

The induction of rdx transcription couples Ci155 processing to Cul3 (Kent, 2006; Zhang, 2006). rdx transcription is regulated by both Hh signaling and Ras signaling, and there were distinctions between cell types. The smo mosaic and hh^ts2 experiments show that Hh signaling is continuously required for rdx transcription in unspecified cells with basal nuclei. In the absence of smo, EGFR-dependent rdx transcription occurs in differentiating photoreceptor cells only, not in unspecified cells. The egfr mosaics show that EGFR is essential for rdx transcription in all cells except the R8 photoreceptor class. Thus, EGFR-dependent differentiation was sufficient to induce rdx in photoreceptors even without Hh signaling, but Hh was not sufficient to induce rdx anywhere without EGFR signaling, except for the R8 cells. Undifferentiated cells might require both the Ras and Hh signaling pathways to induce rdx because the level of Ras signaling is lower in unspecified cells than in differentiating cells of the ommatidia. Alternatively, there may be a combinatorial requirement for both pathways in unspecified cells (Baker, 2009).

There has been some discussion of whether proteolysis of Ci155 by Cul-3 is regulated directly by Hh, as is Cul-1 dependent Ci processing. The current studies provide no support for this idea. In all the genotypes examined, Ci proteolysis correlates with the expression of rdx, and the simplest explanation is that the only effect of Hh on the Cul-3 pathway is through rdx transcription, directly in unspecified cells, and indirectly via EGFR-mediated differentiation in most specified cells (Baker, 2009).

Two mechanisms, acting in different cells, appear to reduce Hh responses through Ci155 after the furrow passes. One also occurs in wing development, where rdx is transcribed only by cells experiencing high Hh signaling levels close to the source of Hh. In wing development, rdx and the Cul3-pathway modulate the amount of Ci155 available for Cul1-dependent processing, lowering the maximum level of Ci155 activity at high Hh levels. Rdx could lower Ci155 levels in unspecified eye cells posterior to the furrow by this mechanism, in which an equilibrium between Hh-dependent induction of rdx, and rdx- and Cul3-dependent degradation of Ci155, leads to a lower level of Ci155 protein than anterior to the furrow. By contrast, in the specified, differentiating eye cells, rdx transcription becomes independent of Hh signaling, and Ci155 is degraded more completely (Baker, 2009).

If there is Hh signaling posterior to the furrow, as these studies find maintains rdx transcription in unspecified retinal cells, why are genes such as atonal that are induced by Hh signaling ahead of the furrow not also expressed posterior to the furrow? There are at least three possible explanations. First, rdx may dampen Ci155 accumulation in unspecified cells such that the threshold necessary for ato expression is not achieved posterior to the furrow. This is unlikely to be the sole explanation, since mutating rdx or cul3 permits Ci155 accumulation but does not lead to ectopic R8 specification, but it could contribute in conjunction with other mechanisms. Secondly, other genes may interfere posterior to the furrow. This could include egfr induction of Bar gene expression, since Bar genes antagonize ato expression. There seem to be multiple respects in which EGFR-dependent differentiation renders cells unable to continue anterior responses to Hh, and it is also envisaged that egfr might play a role in further mechanisms that modulate the response to Dpp signaling posterior to the furrow, should such mechanisms exist. Finally, recent evidence suggests that induction of ato by Hh is not so simple as the induction of a target gene above a threshold in a morphogen gradient, but depends indirectly on Hh repressing Eyeless and activating Sine Oculis, so that these transcription factors are coexpressed and turn on ato only in a domain ahead of the furrow. In this case, persistent Hh signaling would not be expected to activate ato expression once Ey had been repressed (Baker, 2009).

Recently, Hh has been discovered to induce compensatory proliferation in response to eye disc cell death, a further example of post-furrow Hh function. The current results now suggest the model that loss of EGFR-dependent rdx expression elevates Ci155 locally to permit Hh responses when photoreceptor cells that secrete EGFR ligands are lost. Consistent with this idea, loss of rdx or cul3 also result in proliferation of eye disc cells (Baker, 2009).

The regulation of rdx expression and thus degradation of Ci by Cullin-3 may not be sufficient to explain Ci regulation posterior to the furrow. In order for Ci155 to be stable, as observed in cul3 mutant clones and egfr mutant clones, Ci155 must escape processing to Ci75 by Cul-1. Ahead of the furrow, and in most other tissues, rdx is not expressed, Ci is not coupled to Cul3, and Ci155 is stabilized wherever Hh inhibits Smo and the Cul1 pathway. The observation that Ci155 is stable in cul3 clones, or in the genotypes where rdx is not expressed, shows that Ci155 escapes processing by the Cul1 pathway in the posterior eye as well, but this is not due to Hh. Ci155 accumulates in smo egfr mutant clones that do not express rdx and cannot respond to Hh (Baker, 2009).

One model would be that once rdx is induced, Ci155 is sequestered and not available to be processed by Cul1. This model cannot explain why Ci155 accumulates in egfr clones that lack rdx expression, where Ci155 should be available for Cul1. Therefore Ci155 must escape Cul1-mediated processing in the posterior eye by a distinct mechanism. This could be explained by the induction of a component distinct from Rdx that inhibits the processing of Ci155 by Cul1, or sequesters Ci155. It is equally possible that a component essential for processing of Ci155 by Cul1 is repressed posterior to the morphogenetic furrow (Baker, 2009).

Previous studies show that Ci155 never accumulates in smo tkv clones that are unable to respond to either Hh or Dpp signaling. Clones of cells unable to respond to Dpp, but able to respond to Hh and Ras, show only a subtle change in Ci155 labeling. These previously published observations suggest that Ci155 remains a target of Cul1 in the absence of both Dpp and Hh signaling, perhaps through failure to transcribe or repress transcription of a gene that modulates Ci155 proteolysis by Cul1 posterior to the furrow (Baker, 2009).

It is now possible to account for why smo clones affect Ci155 levels differently from cul3 clones, a previously puzzling observation. In cul3 clones, or egfr clones that do not express rdx, the Cul3 pathway cannot degrade Ci155 and the Cul1 pathway is inactivated posterior to the furrow exactly as in wild type discs, so Ci155 accumulates. In smo clones, Ci155 transiently accumulates in those cells in which processing by Cul1 has been lost but rdx not yet induced. In such cells, Ci155 is not coupled to any cullin, and is stable. Eventually, differentiation spreads into the posterior of smo clones, leading to rdx expression, and Cul3-dependent Ci degradation. If differentiation and rdx expression are prevented, as in smo egfr clones, then Ci155 remains stable. Because there is a delay in expressing rdx in smo clones compared to wildtype, Ci155 is not subject to Cul3-mediated processing as soon as in wild type, and there is a period when Ci155 has been uncoupled from Cul1-processing but not yet coupled to the Cul3 pathway. It is during this period that Ci155 accumulates in smo mutant cells (Baker, 2009).

These findings help explain how a wave of differentiation moves across the eye disc uni-directionally. Hh, secreted from differentiating photoreceptor cells, must be present at highest concentrations posterior to the furrow. Indeed, ahead of the furrow Ci155 is stabilized in a decreasing posterior-to-anterior gradient, consistent with a gradient of Hh protein coming from a source posterior to the furrow. Yet, the cell-autonomous responses to Hh signaling that are seen ahead of the furrow, such as cell cycle arrest and atonal expression, do not occur posterior to the furrow, where Ci is rendered unstable by Rdx and Cul3, induced both directly by Hh itself, and indirectly by the photoreceptor differentiation that is largely induced by EGFR posterior to the furrow (Baker, 2009).

There are other examples where Hh-secreting tissues are not the targets of Hh signaling. For, example, in Drosophila wing development, anterior compartments respond to Hh secreted by posterior compartments, but posterior compartment cells do not respond because ci transcription is repressed by the posterior-specific protein Engrailed. In vertebrate development, notochord cells express Shh but the responses seen in the nearby spinal cord are not seen in notochord. Such segregation of Hh-producing cells from fields competent to respond to Hh makes sense, if the purpose of Hh signaling in development is to pattern new body regions. Hh signaling is also deregulated in many tumors. Whether any of these tumors activate Hh signaling by affecting GLI protein stability, or other normal down-regulatory mechanisms, remains to be seen. In any case, mechanisms that render cells unresponsive to Hh by coupling Ci155 to the proteosome might prove useful in the treatment of cancers that depend on Hh signaling (Baker, 2009).

EFFECTS OF MUTATION

gft is identified by seven alleles, all of which are zygotic lethal and dominantly suppress the Gα_s* phenotype. The ability of all gft alleles to modify the Gα_s phenotype indicates that the suppression of ectopic Gα_s signalling is a common property of gft mutations and likely results from a loss of gft function. All gft alleles are zygotic lethal, however, homozygous mutant embryos hatch into first instar larvae. To investigate the strength of each allele the lethal phase was determined of all alleles except gft^P34, which contains a closely linked mutation in Gli (35D3) and gft^GR18, which resides in a translocation background. Homozygous gft^HG39, gft^HG43, gft^PZ06340 or gft^d577 embryos or embryos transheterozygous for each allele over deficiency TE35D-GW15, hatch but die during the second larval instar. Larvae homozygous for gft^B14 survive the second larval instar and die as third instar larvae prior to wandering. These data identify the earliest lethal phase of dCul-3 as the second larval instar (Mistry, 2004).

Precise excision of the P-element insertion in gft^PZ06430 reverted its ability to modify the Gα_s* phenotype, indicating that the P-element insertion suppresses the Gα_s* phenotype and reduces gft function. The P-element insertion in gft^PZ06430 was used to clone gft. Genomic DNA flanking this P-element was used to identify eight overlapping cDNAs from this region (Mistry, 2004).

To verify that gft corresponds to dCul-3, molecular lesions in all gft alleles were sought. gft^B14contains a C to T transversion at nucleotide position 2129, which converts an evolutionarily conserved alanine at position 710 to a valine. l(2)gft^P34 contains an in-frame deletion at nucleotide positions 615-920 that removes 101 amino acids between residues 206 and 307. l(2)gft^GR18 was induced by γ-ray mutagenesis in a T(Y;2); Dp(1;2) background (Y,21-35B1; 35B2-60,Y). This lesion results in the loss of a single nucleotide at position 475, causing a frameshift at amino acid 158 and a premature stop at amino acid 167. l(2)gft^PZ06430 contains a 16kb PZ element (P ry⁺, lacZ ) in the first intron of gft, 228 nucleotides from exon 2. gft^HG39 contains a 5 nucleotide deletion at nucleotide positions 2310-2314 that results in a frameshift at amino acid 747 and a premature stop codon at amino acid 748 that removes the C-terminal 26 amino acids of the protein which comprise 50% of the Cullin motif. The lethality of a dCul-3 allele that lacks 50% of this domain provides strong evidence that the conserved C-terminus is essential for function. The identification of molecular lesions in dCul-3 in five gft alleles provides compelling evidence that gft corresponds to dCul-3. For simplicity, gft is referred to as dCul-3 (Mistry, 2004).

The large maternal contribution of dCul-3 mRNA, together with the larval lethality of dCul-3 alleles suggests that maternal dCul-3 products are sufficient for embryonic development. The requirement of dCul-3 function was examined during embryonic development by creating germ line clones that lack maternal and zygotic dCul-3 contribution using the l(2)gft^PZ6340 and l(2)gft^HG39 alleles. For both alleles, few eggs were recovered. These eggs were approximately half the size of wild-type and contained fused dorsal appendages, a phenotype similar to that of loss of EGF-receptor activity. No discernible embryonic structure interior to the chorion was detected. These data indicate that dCul-3 is required in the maternal germ line during oogenesis for patterning and development of the egg (Mistry, 2004).

Since mutations in dCul-3 have no obvious zygotic phenotype and embryos lacking maternal dCul-3 contribution fail to develop, mitotic clonal analysis was used to study the developmental role of dCul-3. In these studies l(2)gft^PZ06430 and l(2)gft^HG39 were used because these belong to the strongest class of dCul-3 alleles. Clones of these alleles exhibit identical phenotypes that can be grouped into three classes. The first phenotypic class is identified by patterning defects in the wing and notum, including alterations in their shape and size, as well as defects in the formation and position of specific cellular structures such as veins. The second class is identified by ectopic formation of sensory organs as well as defects in sensory organ cell lineage. The third class is identified by defects in cell growth and survival (Mistry, 2004).

Large dCul-3 clones exhibit a number of specific defects in adult wings, including perturbation of the overall shape of the wing and in wing vein position and formation. For example, clones that cover the L3 vein often cause a posterior shift in its position. As L3 runs immediately anterior to the AP boundary, alterations to its position likely reflect modifications to the location of the AP boundary. In addition, clones that cover L3 and other veins are associated with loss of vein tissue . Thus, dCul-3 is required to promote wing growth and patterning as well as wing vein formation, suggesting dCul-3 regulates one or more of the signalling pathways that control these events (Mistry, 2004).

dCul-3 clones in the wing and notum also contain ectopic sensory organ formation. In wild-type wings three campaniform sensillae arise evenly spaced along L3. However, dCul-3 clones that encompass a significant portion of L3 invariably contain more than three and can contain as many as eight campaniform sensillae. Ectopic campaniform sensillae also arise between L2 and the anterior wing margin and ectopic bristles arise distally between L2 and L3. dCul-3 notal clones are associated with significant tufting of both micro- and macro-chaetae. These tufts are made up of ectopic fully formed external sensory organs as well as individual sensory organs that contain multiple shafts within a single socket. The presence of ectopic fully-formed sensory organs suggests dCul-3 helps regulate the initial decision of cells to acquire the neural fate while the presence of multiple shafts within a single socket indicates dCul-3 controls cell-fate decisions in the sensory organ lineage. Reduction in Notch pathway function also promotes ectopic sensory organ formation and shaft duplications, hinting at a possible link between dCul-3 and Notch signalling activity (Mistry, 2004).

This study investigated whether dCul-3 overexpression produces reciprocal wing and notal phenotypes relative to loss of dCul-3 function. Overexpression of full-length dCul-3 (dCul-3FL) along the AP boundary results in decreased intervein territory between L3 and L4, partial or complete loss of the L3 campaniform sensillae and ectopic vein formation. Similarly, overexpression of dCul-3FL in all proneural clusters using scabrous-GAL4 (sca-GAL4) causes a severe loss of macrochaetae throughout the notum as well as campaniform sensillae and anterior margin bristles in the wing. Thus, with respect to vein formation and sensory organ development, overexpression of dCul-3FL results in reciprocal phenotypes to those observed in dCul-3 mutant clones (Mistry, 2004).

Studies in other systems have provided contradictory results on the importance of the Cullin C-terminal domain. Identification of a mutation in the dCul-3 C-terminal domain that severely disrupts dCul-3 function supports the importance of this highly conserved domain. To address this issue further, a truncated form of dCul-3 that specifically lacks the C-terminal domain was driven under the control of sca-GAL4 or dpp-GAL4. Overexpression of this protein has no effect on wing development. These data underline the importance of the C-terminal domain for dCul-3 function (Mistry, 2004).

Perturbation of dCul-3 in proneural clusters affects sensory organ formation. Genetic studies indicate that dCul-3 function opposes sensory organ development. To investigate when dCul-3 exerts its effect on sensory organ development, the expression of neuralized-LacZ (neurLacZ) was followed in mitotic clones of dCul-3 in wing imaginal discs. neurLacZ is expressed in all sensory organ precursors (SOPs) of developing third instar imaginal discs. dCul-3 mitotic clones that overlap areas of SOP formation show a dramatic increase in SOP numbers, while clones that do not overlap areas of SOP formation appear wild-type. Therefore, dCul-3 inhibits SOP development and dCul-3 might normally act either to stabilise proteins that promote sensory organ development, such as Achaete or Scute, or to inhibit proteins that oppose sensory organ development such as members of the Notch pathway (Mistry, 2004).

To investigate if dCul-3 overexpression inhibits SOP formation, Achaete (Ac) and Cut expression was followed in wing imaginal discs in which dCul-3FL was over-expressed using dpp-GAL4 and sca-GAL4. Ac is expressed in proneural clusters and promotes SOP formation while SOPs activate Cut shortly after they form. Overexpression of dCul-3FL leads to the absence or severe reduction of Ac expression in proneural clusters. For example, dCul-3FL overexpression along the AP compartment boundary causes a severe reduction in both Ac and Cut expression in the L3 cluster and a reduction of Ac expression in part of the dorsal component of the anterior wing margin. Overexpression of dCul-3FL in all proneural clusters using sca-Gal4 reduces Ac expression in the anterior wing margin and L3 cluster and also reduces or eliminates Ac expression in most proneural clusters in the notum which in turn results in the loss of SOPs and macrochaetae. These data indicate that dCul-3 affects sensory organ formation by inhibiting Ac expression and thus limiting the ability of cells to acquire the sensory organ precursor fate (Mistry, 2004).

Many of the dCul-3 wing and notal phenotypes are similar to those that arise due to defects in different developmental signalling pathways. Consistent with dCul-3 regulating Hh-pathway activity, dCul-3 clones in third instar eye imaginal discs exhibit a cell-autonomous accumulation of Ci155 posterior to the morphogenetic furrow, but have no effect on Ci155 accumulation anterior to the morphogenetic furrow. Ou (2002) has reported similar observations in dCul-3 eye clones. Because of the importance of targeted proteolysis in the Hh pathway and the roles the Hh pathway plays in setting up the AP boundary of the wing as well as the location of the L3 and L4 veins, whether dCul-3 modulates Hh activity in wing imaginal discs was investigated. Active Hh signalling prevents proteolysis of the full-length form of Ci (Ci155), the transcriptional effector of the Hh pathway. Therefore, an antibody specific to Ci155 was used to assay whether dCul-3 function affects Hh signalling by regulating the stability of Ci155 in larval third instar wing imaginal discs (Mistry, 2004).

Normally, Ci155 is expressed throughout the anterior compartment of the wing disc but accumulates to higher levels in a stripe at the AP boundary in response to high levels of Hh. dCul-3 clones that arise anywhere in the anterior compartment exhibit no clear effect on Ci155 accumulation. Similarly, clones that arise in the posterior compartment and at a distance from the AP boundary have no effect on Ci155. However, dCul-3 clones that reside in the posterior compartment and abut the AP boundary appear to cause a moderate non-autonomous reduction of Ci155 accumulation in cells of the anterior compartment immediately adjacent to the clone. Since high level Ci155 accumulation in these cells normally requires transmission of the Hh signal from posterior compartment cells, these data suggest that dCul-3 activity might modulate transmission of the Hh signal. Thus, in both wing and eye imaginal discs dCul-3 function regulates Ci155 accumulation: in the wing, loss of dCul-3 results in a non-autonomous decrease of Ci155 accumulation, while in the eye, loss of dCul-3 results in a cell autonomous accumulation of Ci155 (Mistry, 2004).

Given the effect loss of dCul-3 function has on Ci155 accumulation, it was asked if dCul-3 overexpression modulates Ci155 accumulation. sca-GAL4 was used to overexpress dCul-3FL in all proneural clusters of the wing imaginal disc and along the entire dorsal-ventral (DV) boundary of the wing margin and a cell-autonomous accumulation of Ci155 was observed in all Sca-expressing cells in the anterior compartment. No effect on Ci155 accumulation in the posterior compartment was observed even though dCul-3FL is overexpressed in the posterior domain of the wing blad. Similarly, dCul-3 overexpression in the leg and haltere, but not eye, results in a cell-autonomous accumulation of Ci155 in the anterior but not posterior compartment. Overexpression of dCul-3 in the wing, leg and haltere imaginal discs using other GAL4 driver lines, results in an identical cell-autonomous accumulation of Ci155 in the anterior compartment. Thus, overexpression of dCul-3FL acts cell-autonomously and specifically in the anterior compartment to promote high-level accumulation of Ci155. These results suggest that dCul-3 directly opposes the action of a protein required to promote Ci cleavage, or that it exhibits dominant negative activity by titrating other SCF components required for Ci proteolysis, such as dCul- (Mistry, 2004).

Costal-2 (Cos-2), Fused (Fu) and Suppressor of Fused (Su(Fu)) form a complex with Ci155 and regulate its cleavage. Antibodies against Cos-2, Fu and Su(Fu) were used to investigate if these proteins are also overexpressed in scaGal4::UASdCul-3FL wing imaginal discs. In contrast to Ci155, expression of all three proteins appears normal in when scaGal4 was used to drive UASdCul-3FL in wing imaginal discs suggesting that overexpression of dCul-3FL specifically targets Ci (Mistry, 2004).

To examine whether persistent dCul-3 overexpression in the posterior compartment might lead to heightened Ci155 accumulation, en-Gal4 was used to overexpress dCul-3FL in this domain. Overexpression of dCul-3FL in the posterior compartment causes a drastic reduction in the size of this compartment and a concomitant increase in the size of the anterior compartment. The drastic size reduction of the posterior compartment along with defects in DV boundary formation in the posterior compartment indicate that overexpression of dCul-3FL leads to defects in cell-survival and cell-fate specification in this domain (Mistry, 2004).

EVOLUTIONARY HOMOLOGS

Identification of mammalian Cul-3 genes

By using a mRNA differential display technique to search for salicylate suppressible genes, a cDNA in human foreskin fibroblasts has been identified that shows sequence homology to the cullin/Cdc53 (CUL) family genes, especially CUL-3. The full-length human CUL-3 (Hs-CUL-3) cDNA has been cloned. It encodes a 768-amino acid polypeptide and has a predicted molecular weight of 88,939. The amino acid sequence of Hs-CUL-3 shows 46% homology to that of its Caenorhabditis elegans ortholog, Ce-CUL-3, and 27% and 23% to that of Hs-CUL-1 and Hs-CUL-2, respectively. Northern blot analysis showed that phorbol 12-myristate 13-acetate increases the expression of Hs-CUL-3 mRNA in a concentration- and time-dependent manner, and this increase is inhibited by sodium salicylate. Hs-CUL-3 is widely expressed in human tissues, and its expression in cultured COLO205 colon cancer cells is increased when compared with that in normal colon cells. It is likely that Hs-CUL-3 is involved in cell proliferation control (Du, 1998).

BTB/POZ domain proteins are substrate adaptors for Cullin 3 ubiquitin ligases

Programmed destruction of regulatory proteins through the ubiquitin-proteasome system is a widely used mechanism for controlling signalling pathways. Cullins are proteins that function as scaffolds for modular ubiquitin ligases typified by the SCF (Skp1-Cul1-F-box) complex. The substrate selectivity of these E3 ligases is dictated by a specificity module that binds cullins. In the SCF complex, this module is composed of Skp1, which binds directly to Cul1, and a member of the F-box family of proteins. F-box proteins bind Skp1 through the F-box motif and substrates by means of carboxy-terminal protein interaction domains. Similarly, Cul2 and Cul5 interact with BC-box-containing specificity factors through the Skp1-like protein elongin C. Cul3 is required for embryonic development in mammals and Caenorhabditis elegans but its specificity module is unknown. A large family of BTB-domain proteins has been identified as substrate-specific adaptors for C. elegans CUL-3. Biochemical studies using the BTB protein MEL-26 (Drosophila homolog: CG9924) and its genetic target MEI-1 (Drosophila homolog: katanin 60) indicate that BTB proteins merge the functional properties of Skp1 and F-box proteins into a single polypeptide (Xu, 2003).

Cullins (CULs) are subunits of a prominent class of RING ubiquitin ligases. Whereas the subunits and substrates of CUL1-associated SCF complexes and CUL2 ubiquitin ligases are well established, they are largely unknown for other cullin family members. S. pombe CUL3 (Pcu3p) forms a complex with the RING protein Pip1p and all three BTB/POZ domain proteins encoded in the fission yeast genome. The integrity of the BTB/POZ domain, which shows similarity to the cullin binding proteins SKP1 and elongin C, is required for this interaction. Whereas Btb1p and Btb2p are stable proteins, Btb3p is ubiquitylated and degraded in a Pcu3p-dependent manner. Btb3p degradation requires its binding to a conserved N-terminal region of Pcu3p that precisely maps to the equivalent SKP1/F box adaptor binding domain of CUL1. It is proposed that the BTB/POZ domain defines a recognition motif for the assembly of substrate-specific RING/cullin 3/BTB ubiquitin ligase complexes (Geyer, 2003).

These results identified BTB/POZ proteins as components of Pcu3p/Pip1p ubiquitin ligase complexes. Four pieces of evidence suggest that BTB/POZ domain proteins are functionally equivalent to the SKP1/F box adaptor dimers determining the substrate specificity of CUL1-associate SCF complexes: (1) all three BTB/POZ proteins present in the fission yeast genome interact with Pcu3p/Pip1p complexes; (2) BTB/POZ domains are structurally related to SKP1; (3) N-terminal residues invariably conserved in all CUL3 homologs, including Pcu3p, cluster in the same region of CUL1 that mediates its interaction with SKP1/F box adaptor dimers. Both the Btb3p/Pcu3p interaction and Pcu3p-dependent Btb3p degradation depend on the integrity of this conserved N-terminal region. (4) Btb3p is ubiquitylated in vitro in a Pcu3p-dependent manner, a finding reminiscent of CUL1-dependent ubiquitylation and degradation of F box proteins. Taken together, these findings strongly suggest that the BTB/POZ domain proteins ubiquitously present in eukaryotes define a family of substrate-specific adaptors for CUL3. Since fission yeast encodes three different BTB/POZ domain proteins, all of which interact with Pcu3p and Pip1p, it may form a minimum of three distinct RING/cullin 3/BTB complexes (Geyer, 2003).

The concentrations and functions of many cellular proteins are regulated by the ubiquitin pathway. Cullin family proteins bind with the RING-finger protein Roc1 to recruit the ubiquitin-conjugating enzyme (E2) to the ubiquitin ligase complex (E3). Cul1 and Cul7, but not other cullins, bind to an adaptor protein, Skp1. Cul1 associates with one of many F-box proteins through Skp1 to assemble various SCF-Roc1 E3 ligases that each selectively ubiquitinate one or more specific substrates. Cul3, but not other cullins, binds directly to multiple BTB domains through a conserved amino-terminal domain. In vitro, Cul3 promotes ubiquitination of Caenorhabditis elegans MEI-1, a katanin-like protein whose degradation requires the function of both Cul3 and BTB protein MEL-26. It is suggested that in vivo there exists a potentially large number of BCR3 (BTB-Cul3-Roc1) E3 ubiquitin ligases (Furukawa, 2003).

CUL-3 function in C. elegans: CUL-3 is regulated by nedulation and MEL-26 is a substrate-specific adaptor of the CUL-3 based ubiquitin-ligase complex

SCF (Skp1-Cullin-F-box) complexes are a major class of E3 ligases that are required to selectively target substrates for ubiquitin-dependent degradation by the 26S proteasome. Conjugation of the ubiquitin-like protein Nedd8 to the cullin subunit (neddylation) positively regulates activity of SCF complexes, most likely by increasing their affinity for the E2 conjugated to ubiquitin. The Nedd8 conjugation pathway is required in C. elegans embryos for the ubiquitin-mediated degradation of the microtubule-severing protein MEI-1/Katanin at the meiosis-to-mitosis transition. Genetic experiments suggest that this pathway controls the activity of a CUL-3-based E3 ligase. Counteracting the Nedd8 pathway, the COP9/signalosome has been shown to promote deneddylation of the cullin subunit. However, little is known about the role of neddylation and deneddylation for E3 ligase activity in vivo. The COP9/signalosome has been identified and characterized in C. elegans; it promotes deneddylation of CUL-3, a critical target of the Nedd8 conjugation pathway. As in other species, the C. elegans signalosome is a macromolecular complex containing at least six subunits that localizes in the nucleus and the cytoplasm. Reducing COP9/signalosome function by RNAi results in a failure to degrade MEI-1, leading to severe defects in microtubule-dependent processes during the first mitotic division. Intriguingly, reducing COP9/signalosome function suppresses a partial defect in the neddylation pathway; this suppression suggests that deneddylation and neddylation antagonize each other. It is concluded that both neddylation and deneddylation of CUL-3 is required for MEI-1 degradation and it is proposed that cycles of CUL-3 neddylation and deneddylation are necessary for its ligase activity in vivo (Pintard, 2003a).

Many biological processes, such as development and cell cycle progression are tightly controlled by selective ubiquitin-dependent degradation of key substrates. In this pathway, the E3-ligase recognizes the substrate and targets it for degradation by the 26S proteasome. The SCF (Skp1-Cul1-F-box) and ECS (Elongin C-Cul2-SOCS box) complexes are two well-defined cullin-based E3-ligases. The cullin subunits serve a scaffolding function and interact through their C terminus with the RING-finger-containing protein Hrt1/Roc1/Rbx1, and through their N terminus with Skp1 or Elongin C, respectively. In Caenorhabditis elegans, the ubiquitin-ligase activity of the CUL-3 complex is required for degradation of the microtubule-severing protein MEI-1/katanin at the meiosis-to-mitosis transition. However, the molecular composition of this cullin-based E3-ligase is not known. The BTB-containing protein MEL-26 has been identified as a component required for degradation of MEI-1 in vivo. Importantly, MEL-26 specifically interacts with CUL-3 and MEI-1 in vivo and in vitro, and displays properties of a substrate-specific adaptor. These results suggest that BTB-containing proteins may generally function as substrate-specific adaptors in Cul3-based E3-ubiquitin ligases (Pintard, 2003b).

Neddylation and deneddylation regulate Cul1 and Cul3 protein accumulation

Cullin family proteins organize ubiquitin ligase (E3) complexes to target numerous cellular proteins for proteasomal degradation. Neddylation, the process that conjugates the ubiquitin-like polypeptide Nedd8 to the conserved lysines of cullins, is essential for in vivo cullin-organized E3 activities. Deneddylation, which removes the Nedd8 moiety, requires the isopeptidase activity of the COP9 signalosome (CSN). This study shows that in cells deficient for CSN activity, cullin1 (Cul1) and cullin3 (Cul3) proteins are unstable, and that to preserve their normal cellular levels, CSN isopeptidase activity is required. It is further shown that neddylated Cul1 and Cul3 are unstable - as suggested by the evidence that Nedd8 promotes the instability of both cullins - and that the unneddylatable forms of cullins are stable. The protein stability of Nedd8 is also subject to CSN regulation and this regulation depends on its cullin-conjugating ability, suggesting that Nedd8-conjugated cullins are degraded en bloc. It is proposed that while Nedd8 promotes cullin activation through neddylation, neddylation also renders cullins unstable. Thus, CSN deneddylation recycles the unstable, neddylated cullins into stable, unneddylated ones, and promotes cullin-organized E3 activity in vivo (Wu, 2005).

Targets of Cullin 3-Roc1 ligases

Cyclin E is an unstable protein that is degraded in a ubiquitin- and proteasome- dependent pathway. Two factors stimulate cyclin E ubiquitination in vivo: when it is free of its CDK partner, and when it is phosphorylated on threonine 380. The first of these pathways was pursued by using a two-hybrid screen to identify proteins that could bind only to free cyclin E. This resulted in the isolation of human Cul-3, a member of the cullin family of E3 ubiquitin-protein ligases. Cul-3 is bound to cyclin E but not to cyclin E-Cdk2 complexes in mammalian cells, and overexpression of Cul-3 increases ubiquitination of cyclin E but not other cyclins. Conversely, deletion of the Cul-3 gene in mice causes increased accumulation of cyclin E protein, and has cell-type-specific effects on S-phase regulation. In the extraembryonic ectoderm, in which cells undergo a standard mitotic cycle, there is a greatly increased number of cells in S phase. In the trophectoderm, in which cells go through endocycles, there is a block to entry into S phase. The SCF pathway, which targets cyclins for ubiquitination on the basis of their phosphorylation state, and the Cul-3 pathway, which selects cyclin E for ubiquitination on the basis of its assembly into CDK complexes, may be complementary ways to control cyclin abundance (Singer, 1999).

DNA topoisomerase I (TOP1)-DNA covalent complexes are the initial lesions produced by antitumor camptothecins (CPTs). The TOP1-directed drugs stimulate degradation of TOP1 via the ubiquitin-proteasome pathway. Proteasome inhibition prevents degradation of DNA-bound TOP1 and sustains high levels of covalent complexes, thus enhancing CPT-induced cell death. Consistent with this, increased degradation of TOP1-DNA covalent complexes was seen in acquired CPT-resistant cells. The resistant cells showed elevated expressions of Cul3, a member of the cullin family of E3 ubiquitin ligases. The reduction in Cul3 expression by small interfering RNA decreased degradation of TOP1-DNA covalent complexes. Conversely, Cul3 overexpression by stable transfection promoted covalent complex degradation and reduced CPT-induced cell death without affecting basal TOP1 expression levels. These results indicate that Cul3, by promoting proteasomal degradation of TOP1-DNA covalent complexes, becomes an important regulator for cellular CPT sensitivity (Zhang, 2004).

Transcription factor Nrf2 is a major regulator of genes encoding phase 2 detoxifying enzymes and antioxidant stress proteins in response to electrophilic agents and oxidative stress. In the absence of such stimuli, Nrf2 is inactive owing to its cytoplasmic retention by Keap1 and rapid degradation through the proteasome system. The contribution of Keap1 to the rapid turnover of Nrf2 (half-life of less than 20 min) was examined, and it was found that a direct association between Keap1 and Nrf2 is required for Nrf2 degradation. In a series of domain function analyses of Keap1, it was found that both the BTB and intervening-region (IVR) domains are crucial for Nrf2 degradation, implying that these two domains act to recruit ubiquitin-proteasome factors. Indeed, Cullin 3 (Cul3), a subunit of the E3 ligase complex, was found to interact specifically with Keap1 in vivo. Keap1 associates with the N-terminal region of Cul3 through the IVR domain and promotes the ubiquitination of Nrf2 in cooperation with the Cul3-Roc1 complex. These results thus provide solid evidence that Keap1 functions as an adaptor of Cul3-based E3 ligase. Nrf2 and Keap1 are the first reported mammalian substrate and adaptor, respectively, of the Cul3-based E3 ligase system (Kobayashi, 2004).

The concentrations and functions of many eukaryotic proteins are regulated by the ubiquitin pathway, which consists of ubiquitin activation (E1), conjugation (E2), and ligation (E3). Cullins are a family of evolutionarily conserved proteins that assemble by far the largest family of E3 ligase complexes. Cullins, via a conserved C-terminal domain, bind with the RING finger protein Roc1 to recruit the catalytic function of E2. Via a distinct N-terminal domain, individual cullins bind to a protein motif present in multiple proteins to recruit specific substrates. Cullin 3 (Cul3), but not other cullins, binds directly with BTB domains to constitute a potentially large number of BTB-CUL3-ROC1 E3 ubiquitin ligases. The human BTB-Kelch protein Keap1, a negative regulator of the antioxidative transcription factor Nrf2, binds to CUL3 and Nrf2 via its BTB and Kelch domains, respectively. The KEAP1-CUL3-ROC1 complex promotes NRF2 ubiquitination in vitro; knocking down Keap1 or CUL3 by short interfering RNA results in NRF2 protein accumulation in vivo. It is suggested that Keap1 negatively regulates Nrf2 function in part by targeting Nrf2 for ubiquitination by the CUL3-ROC1 ligase and subsequent degradation by the proteasome. Blocking NRF2 degradation in cells expressing both KEAP1 and NRF2 by either inhibiting the proteasome activity or knocking down Cul3, results in NRF2 accumulation in the cytoplasm. These results may reconcile previously observed cytoplasmic sequestration of NRF2 by KEAP1 and suggest a possible regulatory step between KEAP1-NRF2 binding and NRF2 degradation (Furukawa, 2005).

Dishevelled is a conserved protein that interprets signals received by Frizzled receptors. Using a tandem-affinity purification strategy and mass spectrometry proteins have been identified associated with Dishevelled, including a Cullin-3 ubiquitin ligase complex containing the BTB protein Kelch-like 12 (KLHL12). This E3 ubiquitin ligase complex is recruited to Dishevelled in a Wnt-dependent manner that promotes its poly-ubiquitination and degradation. Functional analyses demonstrate that regulation of Dishevelled by this ubiquitin ligase antagonizes the Wnt-beta-catenin pathway in cultured cells, as well as in Xenopus and zebrafish embryos. Considered with evidence that the distinct Cullin-1 based SCF(beta-TrCP)complex regulates beta-catenin stability, these data on the stability of Dishevelled demonstrates that two distinct ubiquitin ligase complexes regulate the Wnt-beta-catenin pathway (Angers, 2006).

A Cul3-based E3 ligase removes Aurora B from mitotic chromosomes, regulating mitotic progression and completion of cytokinesis in human cells

Faithful cell-cycle progression is tightly controlled by the ubiquitin-proteasome system. A human Cullin 3-based E3 ligase (Cul3) has been identified that is essential for mitotic division. In a complex with the substrate-specific adaptors KLHL9 and KLHL13, Cul3 is required for correct chromosome alignment in metaphase, proper midzone and midbody formation, and completion of cytokinesis. This Cul3-based E3 ligase removes components of the chromosomal passenger complex from mitotic chromosomes and allows their accumulation on the central spindle during anaphase. Aurora B directly binds to the substrate-recognition domain of KLHL9 and KLHL13 in vitro, and coimmunoprecipitates with the Cul3 complex during mitosis. Moreover, Aurora B is ubiquitylated in a Cul3-dependent manner in vivo, and by reconstituted Cul3/KLHL9/KLHL13 ligase in vitro. It is thus proposed that the Cul3/KLHL9/KLHL13 E3 ligase controls the dynamic behavior of Aurora B on mitotic chromosomes, and thereby coordinates faithful mitotic progression and completion of cytokinesis (Sumara, 2007).

Golgi-associated RhoBTB3 targets Cyclin E for ubiquitylation and promotes cell cycle progression

Cyclin E regulates the cell cycle transition from G1 to S phase and is degraded before entry into G2 phase. This study shows that in mice RhoBTB3, a Golgi-associated, Rho-related ATPase, regulates the S/G2 transition of the cell cycle by targeting Cyclin E for ubiquitylation. Depletion of RhoBTB3 arrested cells in S phase, triggered Golgi fragmentation, and elevated Cyclin E levels. On the Golgi, RhoBTB3 bound Cyclin E as part of a Cullin3 (CUL3)-dependent RING-E3 ubiquitin ligase complex comprised of RhoBTB3, CUL3, and RBX1. Golgi association of this complex is required for its ability to catalyze Cyclin E ubiquitylation and allow normal cell cycle progression. These experiments reveal a novel role for a Ras superfamily member in catalyzing Cyclin E turnover during S phase, as well as an unexpected, essential role for the Golgi as a ubiquitylation platform for cell cycle control (Lu, 2013).

The ID1-CULLIN3 axis regulates intracellular SHH and WNT signaling in glioblastoma stem cells

Inhibitor of differentiation 1 (ID1; see Drosophila Extra macrochaetae) is highly expressed in glioblastoma stem cells (GSCs). However, the regulatory mechanism responsible for its role in GSCs is poorly understood. This study reports that ID1 activates GSC proliferation, self-renewal, and tumorigenicity by suppressing CULLIN3 ubiquitin ligase (see Drosophila Cullin-3). ID1 induces cell proliferation through increase of CYCLIN E, a target molecule of CULLIN3. ID1 overexpression or CULLIN3 knockdown confers GSC features and tumorigenicity to murine Ink4a/Arf-deficient astrocytes. Proteomics analysis revealed that CULLIN3 interacts with GLI2 (see Drosophila Cubitus interruptus) and DVL2 (see Drosophila Dishevelled) and induces their degradation via ubiquitination. Consistent with ID1 knockdown or CULLIN3 overexpression in human GSCs, pharmacologically combined control of GLI2 and beta-CATENIN (see Drosophila Armadillo) effectively diminishes GSC properties. A ID1-high/CULLIN3-low expression signature correlates with a poor patient prognosis, supporting the clinical relevance of this signaling axis. Taken together, a loss of CULLIN3 represents a common signaling node for controlling the activity of intracellular WNT and SHH signaling pathways mediated by ID1.

REFERENCES

Search PubMed for articles about Drosophila Cullin-3

Angers, S., et al. (2006). The KLHL12-Cullin-3 ubiquitin ligase negatively regulates the Wnt-β-catenin pathway by targeting Dishevelled for degradation. Nat. Cell Biol. 8: 348-357. 16547521

Arama, E., Bader, M., Rieckhof, G.E., and Steller, H. (2007). A ubiquitin ligase complex regulates caspase activation during sperm differentiation in Drosophila. PLoS Biol. 5: e251. PubMed Citation: 17880263

Baker, N. E., Bhattacharya, A. and Firth, L. C. (2009). Regulation of Hh signal transduction as Drosophila eye differentiation progresses. Dev. Biol. 335(2): 356-66. PubMed Citation: 19761763

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date revised: 22 February 2022

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