The Cul1-based SCFSlimb complex controls CiFL processing in the anterior cells of the eye disc. In contrast, 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 or 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).
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 hhts2 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).
The genetic evidence suggests that Nedd8 is directly required for CiFL proteolytic processing, consistent with the hypothesis that neddylation affects CiFL proteolysis through regulating SCFSlimb activity. Cullin proteins are the identified targets for Nedd8 modification. In the Drosophila genome, six Cullin proteins are identified, each corresponding to a mammalian homolog. Among them, Cul1 is involved in the formation of SCF complexes that function as E3 ligase. The null Cul1 allele, Cul1EX was generated. In Cul1EX homozygous larvae in the first instar stage, the Cul1 signal detected by anti-Cul1 antibodies is almost completely absent. The residual Cul1 protein in Cul1EX larvae is probably maternally contributed (Ou, 2002).
Whether Nedd8 conjugates to Cul1 in Drosophila was investigated further. Cell extracts of eye-antennal discs and brain lobes isolated from wild-type third instar larvae were immunoprecipitated by anti-Cul1 antibodies and immunoblotted by anti-Nedd8 antibodies. The Nedd8-positive signal suggests that Cul1 is modified by Nedd8 in Drosophila. Consistently, the Nedd8-modified Cul1 signal is absent in cell extracts prepared from the first instar Nedd8 mutant larvae (Ou, 2002).
In addition, it was found that unmodified Cul1 accumulates in the Nedd8 hypomorphic mutants Nedd8EP(2)2063/AN015. This effect of Cul1 accumulation is also observed in Nedd8 null mutant clones in third-instar eye discs. These data suggested that Nedd8 modification of Cul1 might modulate Cul1 stability (Ou, 2002).
To test whether Nedd8 activity in controlling protein stability is mediated through Cul1, whether depleting Cul1 would exhibit the same phenotypes as depleting Nedd8 was examined. Cul1EX clones were generated in developing wing discs, and the protein levels of CiFL and Arm were examined. Accumulations of CiFL and Arm were found in Cul1EX mutant cells, identical to the phenotypes observed in Nedd8 mutant clones. In the eye disc, CycE accumulates in Cul1 mutant cells. These results suggest that Nedd8 and Cul1 function together for controlling the protein stability of Ci and Arm in the wing disc and CycE in the eye disc (Ou, 2002).
Whether the Cul1-based SCF complex is involved in controlling CiFL stability in the eye disc was examined. When located anterior to the MF, Cul1EX cells accumulate high levels of CiFL, a phenotype similar to Nedd8 mutant cells. However, Cul1EX cells that located posterior to the MF only expressed a basal level of CiFL, suggesting that the Cul1-based SCF complex may not control Ci stability in the posterior cells (Ou, 2002).
In addition to the Nedd8-Cul1 core component, the SCF complex also includes a substrate-specific F-box protein. To investigate whether SCF activity in CiFL processing is limited to the anterior cells of the eye disc, the mutant phenotype of slimb was examined. slimb is required for CiFL proteolytic processing in tissues such as wing and leg discs. When slimb1 mutant clones were generated in eye discs, high levels of CiFL accumulation were detected exclusively in clones located anterior to the MF. No accumulation of CiFL could be detected in posterior slimb1 clones. Suppression of CiFL accumulation in the posterior cells was not due to possible residual activity present in hypomorphic slimb1, because identical results of CiFL accumulation were observed in the strong hypomorphic allele slimb2 and the null allele slimbP (Ou, 2002).
In summary, the results strongly suggested that in vivo, the Nedd8-modified, Cul1-based SCFSlimb complex controls CiFL proteolysis in anterior cells. Following the sweep of the MF, CiFL stability in the posterior cells is controlled by an SCFSlimb-independent mechanism (Ou, 2002).
The inducible production of antimicrobial peptides is a major immune response in Drosophila. The genes encoding these peptides are activated by NF-κB transcription factors that are controlled by two independent signaling cascades: the Toll pathway that regulates the NF-κB homologs, Dorsal and DIF; and the IMD pathway that regulates the compound NF-κB-like protein, Relish. Although numerous components of each pathway that are required to induce antimicrobial gene expression have been identified, less is known about the mechanisms that either repress antimicrobial genes in the absence of infection or that downregulate these genes after infection. In a screen for factors that negatively regulate the IMD pathway, two partial loss-of-function mutations were isolated in the SkpA gene that constitutively induce the antibacterial peptide gene, Diptericin, a target of the IMD pathway. These mutations do not affect the systemic expression of the antifungal peptide gene, Drosomycin, a target of the Toll pathway. SkpA encodes a homolog of the yeast and human Skp1 proteins. Skp1 proteins function as subunits of SCF-E3 ubiquitin ligases that target substrates to the 26S proteasome, and mutations affecting either the Drosophila SCF components, Slimb and Cullin1, or the proteasome also induce Diptericin expression. In cultured cells, inhibition of SkpA and Slimb via RNAi increases levels of both the full-length Relish protein and the processed Rel-homology domain. It is concluded that in contrast to other NF-κB activation pathways, the Drosophila IMD pathway is repressed by the ubiquitin-proteasome system. A possible target of this proteolytic activity is the Relish transcription factor, suggesting a mechanism for NF-κB downregulation in Drosophila (Khush, 2002).
In wild-type flies Diptericin is tightly controlled by the IMD pathway. Therefore, to identify genes that normally function to repress the IMD pathway, 2,000 yellow, white (y,w) F1 male progeny from male flies mutagenized with ethyl methanesulfonate were screened for constitutive expression of a Green Fluorescent Protein (GFP) reporter gene under the control of the Diptericin promoter. Two males, J6 and G49, expressed Diptericin-GFP, and this gene was constitutively expressed in larvae and adults in homozygous lines derived from these males. Although flies carrying the J6 and G49 mutations are viable and fertile at 25°C, G49 is pupal lethal at 29°C, indicating temperature-sensitive phenotypes associated with this mutation (Khush, 2002).
Using recombination mapping, the J6 and G49 mutations were shown to be tightly linked to the y locus on the proximal tip of the X chromosome. To further localize the two mutations, deletions were used to determine that J6 falls in the area defined by the overlap of Df(1)74k24.1, Df(1)svr, and Df(1)su(s)83, placing it in cytological region 1B10 near the Dredd gene. Two lethal P-element insertions in the Bloomington stock center collection, l(1)G0389 and l(1)G0109, which map near this region, were shown to not complement the constitutive Diptericin expression in the J6 and G49 lines. By sequencing DNA flanking the P elements in the two insertion lines, both elements were ascertained to lie within 200 bp of each other in the 5' untranslated region of the SkpA gene. To confirm that J6 and G49 are mutations in SkpA, a wild-type SkpA transgene on the second chromosome was shown to suppress the constitutive Diptericin expression phenotype in G49 flies. The J6 and G49 lines were shown to each contain a point mutation in the SkpA gene that generates a single amino acid change in the SkpA protein: J6, renamed SkpAJ6, converts threonine 98 to an isoleucine, and G49, renamed SkpAG49, replaces glutamic acid 101 with a lysine. These alleles are hypomorphic mutations of SkpA since the P-element insertions are pupal lethal at 25°C. SkpAG49 is pupal lethal at 29°C, and homozygous SkpAG49 adults transferred to 29°C express Diptericin at similar levels as flies heterozygous for SkpAG49 and either the P-element insertions or deletions that remove SkpA. At 29°C, therefore, SkpAG49 behaves like a null mutation, which probably reflects the significant change from the negatively charged glutamic acid to the positively charged lysine in this allele (Khush, 2002).
The SkpA gene encodes a protein that is highly similar to Skp1 proteins in humans and yeast. Skp1 proteins are components of SCF ubiquitin ligases that target substrates to the proteasome, and crystal structures of human Skp1 complexed with the F-box protein Skp2 and the cullin protin Cul1 have been solved. SkpAJ6 and SkpAG49 both affect a conserved region of SkpA that corresponds to helix 5 of Skp1; helix 5 forms part of the core interface between Skp1, the F-box region of Skp2, and the amino-terminal domain of Cul1, with some amino acids in this helix making direct contact with residues in Skp2 and Cul1. This suggests that the SkpAJ6 and SkpAG49 mutations disrupt interactions between SkpA and the F-box protein and cullin components of an SCF complex. Protein interaction studies indicate that SkpA functions with the F-box protein Slimb and the Cullin-like protein Cullin1 (Cul1) in a Drosophila SCF complex. In support of this model, slimb1 and dcul1l(2)02074 mutant larvae, as well as larvae carrying the DTS5 mutation, a dominant-negative mutation that affects the β6 subunit of the 26S proteasome, were shown to express Diptericin at levels comparable to those in the SkpA mutants. To further test the DTS5 phenotype, the UAS-Gal4 system was used to overexpress a UAS-DTS5 transgene in larval fat bodies: DTS5 overexpression induces Diptericin to levels that are comparable to those generated by bacterial infection with Erwinia carotovora carotovora 15 (Ecc15). Flies heterozygous for mutations at both the SkpA and slimb loci were generated: these flies constitutively express Diptericin, indicating a synergistic interaction between SkpA and slimb. The constitutive Diptericin expression in the slimb1, dcul1l(2)02074, and DTS5 mutants and the interaction between SkpA and slimb together suggest that an SCFSkpA/Cul1/Slimb ubiquitin ligase represses Diptericin expression by targeting a regulatory factor for degradation by the 26S proteasome (Khush, 2002).
To determine if the constitutive Diptericin expression in the SCF complex mutants is mediated through the IMD pathway, Diptericin levels were examined in larvae homozygous for mutations in either SkpA, or slimb and various genes of the IMD pathway: SkpAJ6;imd1 and SkpAG49;dtak11 double mutants display constitutive Diptericin expression, although Diptericin levels are slightly reduced in the SkpAG49;dtak11 larvae. Mutations in DmIkkγ, DmIkkβ, and Relish, however, completely block Diptericin expression in the SkpAJ6 background, and a Dredd mutation completely blocks Diptericin expression in the slimb1 background. The constitutive Diptericin expression observed in SkpA and slimb mutants, therefore, does not require IMD and dTak1, but it is dependent on the DmIKK complex, Dredd, and Relish. These results imply that, in wild-type flies, the SCFSkpA/Cul1/Slimb negatively regulates the IMD pathway by targeting one of these factors, or an additional unidentified component of the IMD pathway, for degradation by the proteasome. In contrast to fat body cells, the IMD pathway is the primary regulator of all antimicrobial genes, including Drosomycin, in surface epithelial tissues. A Drosomycin-GFP transgene is constitutively expressed in tracheal cells but not in fat body cells of slimb1 mutant larvae; this expression pattern further demonstrates that the IMD pathway, but not the Toll pathway, is constitutively activated when the SCFSkpA/Cul1/Slimb complex is compromised (Khush, 2002).
Although the genetic results do not allow differentiation between the DmIKK complex, Dredd, Relish, or other unidentified downstream components of the IMD pathway as targets of the ubiquitin-proteasome pathway, the mammalian Relish homolog, P105, is regulated by an SCF complex that contains the Slimb homolog β-TrCP/E3RSIκB. Consequently, RNA-mediated interference (RNAi), an effective technique for specifically inhibiting targeted proteins, was used in cultured Drosophila S2 cells to test for interactions between the SCFSkpA/Cul1/Slimb complex and Relish. Initially, SkpA and Slimb activity were blocked in S2 cells via RNAi; then, transient expression of a full-length Relish protein, modified by an N-terminal FLAG tag, was induced in the same S2 cells and the effects of the SkpA and slimb RNAi treatments on FLAG-Relish protein stability was monitored using Western blots and anti-FLAG antibodies (Khush, 2002).
Reducing Slimb activity, in the absence of LPS stimulation, visibly increases steady-state levels of both full-length Relish and the active N-terminal Rel-homology domain; levels of both polypeptides are further increased by inhibiting Slimb and SkpA simultaneously. This effect is specific since RNAi of the SkpA homologs, SkpB and SkpD, does not increase Relish levels. Dredd RNAi does increase Relish levels at day 1, but this is probably because Dredd inhibition blocks Relish processing. Previous studies show that Relish processing in S2 cells is induced by lipopolysaccharide (LPS) and requires Dredd activity. As expected, therefore, RNAi of Dredd blocks LPS-induced Relish processing. Simultaneous RNAi of SkpA and Slimb in the presence of LPS, however, results in higher steady-state levels of the Rel-homology domain up to 4 days after Relish induction. Higher levels of the Rel-homology domain after SkpA and Slimb RNAi could be caused by increased processing of full-length Relish. However, because full-length Relish levels also mount, the explanation is favored that Rel-homology domain turnover is reduced. Although the Slimb and SkpA RNAi treatments appear to inhibit Relish turnover, Relish levels do eventually diminish. This suggests that RNAi efficiency decreases with time, possibly due to degradation of the transfected double-stranded RNA. These RNAi experiments indicate that the constitutive antimicrobial gene expression in SkpA and slimb mutant flies is caused by higher Relish levels, and they suggest that the SCFSkpA/Cul1/Slimb complex represses the IMD pathway by promoting the degradation of both full-length and processed Relish proteins (Khush, 2002).
If the constitutive antimicrobial gene expression in flies carrying mutations that affect the SCFSkpA/Cul1/Slimb complex or proteasome is due to higher Relish levels, this would imply some level of steady-state Relish activation. Low levels of the Rel-homology domain have been reported in nuclear extracts from unstimulated S2 cells, and these low levels indicate that Relish is constitutively processed. Increasing Relish levels in larvae and adults via the Gal4-UAS system is sufficient to induce low levels of Diptericin expression. These results indicate that Relish is constitutively processed and activated to some level, supporting the hypothesis that Relish activity, in the absence of infection, is countered by ubiquitination and degradation (Khush, 2002).
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date revised: 10 February 2010
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