Many proteins are targeted to proteasome degradation by a family of E3 ubiquitin ligases, termed SCF complexes, that link substrate proteins to an E2 ubiquitin-conjugating enzyme. SCFs are composed of three core proteins-Skp1, Cdc53/Cull, Rbx1/Hrt1-and a substrate specific F-box protein. The closest homologs to the human components of the SCF(betaTrCP) complex and the E2 ubiquitin-conjugating enzyme UbcH5 have been identified in Drosophila. Putative Drosophila SCF core subunits skpA and Rbx1 both interact directly with Cu11 and the F-box protein Slimb. The direct interaction of UbcH5 related protein UbcD1 with Cul1 and Slimb is also reported. In addition, a functional complementation test performed on a Saccharomyces cerevisiae Hrt1p-deficient mutant shows that Drosophila Rbx1 is able to restore the yeast cells viability. These results suggest that Rbx1, SkpA, Cullin1, and Slimb proteins are components of a Drosophila SCF complex that functions in combination with the ubiquitin conjugating enzyme UbcD1 (Bocca, 2001).
Nedd8, the ubiquitin-like protein that covalently modifies members of the Cullin family, is highly conserved from yeast to mammals. Several Nedd8 alleles have been identifed in Drosophila, including two null alleles Nedd8AN015 and Nedd8AN024. The Nedd8 null mutants were growth-arrested in the first-instar larval stage and died within several days without further growth. Mutant clones were generated to analyze Nedd8 loss-of-function phenotypes; in the adult flies very few Nedd8AN015 mutant cells are identified, while in control experiments, large Nedd8+ clones are frequently recovered. Nedd8 mutant clones of small size, however, are present in the developing discs, suggesting that Nedd8 mutant cells are defective in proliferation and survival (Ou, 2002).
To study the relationship between Nedd8 and the F-box protein Slimb-mediated protein degradation, the protein stability for substrates of Slimb was studied in Nedd8 mutant cells. Nedd8 mutant cells in developing wing discs accumulate high levels of full-length Ci (CiFL) and Arm proteins, phenotypes identical to those observed in the slimb mutants. In Drosophila embryonic development, the signaling pathway mediated by the NFkappaB homolog Dorsal is required for patterning the dorsoventral identity. Accumulation of pIkappaBa/Cactus inhibits Dorsal activation, leading to repression of the downstream target gene, twist, an effect that has been observed in slimb mutants. twist expression was examined in embryos laid by Nedd8AN015/Nedd8203 females in which Nedd8203 is a hypomorphic allele. In such embryos, the twist expression domain is reduced along the dorsoventral axis and often found missing in many cells, revealing a requirement for Nedd8 in Dorsal signaling (Ou, 2002).
Whether Nedd8 affects the protein level of CycE was examined. Levels of CycE are regulated by the F-box protein, Archipelago (Ago; Moberg, 2001). CycE accumulates in Nedd8 mutant cells in the eye disc. These results suggest that Nedd8 might affect the stability of a broad range of proteins through F-box proteins in flies (Ou, 2002).
The Drosophila eye imaginal disc is an excellent model system for developmental study. Cells are undifferentiated and dividing randomly anterior to the MF, and cells posterior to the MF are differentiating into different types of cells. Thus, Nedd8 phenotypes can be observed in cells of different differentiation states in a single eye disc. The Hh pathway is the major signaling pathway in eye development, and the protein level of its effector Ci is tightly regulated in Drosophila. These studies focused on how Nedd8 regulates the CiFL level in the Hh pathway and the effects of Ci upregulation on eye development. It was found that in the Nedd8 clones that located anterior to the MF, CiFL accumulates to a level identical to that in the MF cells that transduce the Hh signaling pathway. Accumulation of CiFL also exists in posterior mutant cells that locate proximally but not distally to the MF. CiFL accumulation in Nedd8 mutant cells is not caused by an increase in the ci transcription level, because expression of ci-lacZ that recapitulates endogenous ci expression remains constant in Nedd8 mutant cells, indicating that posttranscriptional defects resulted in CiFL accumulation (Ou, 2002).
Elevated CiFL levels causes anterior Nedd8 mutant cells to adopt MF fate precociously. Nedd8 mutant cells are constricted on their apical surface, as revealed by the intensified phalloidin staining, and express the Hh-target gene, dpp, as detected by the expression of dpp-lacZ reporter gene. Furthermore, the early photoreceptor marker, Atonal, is induced. These phenotypes are observed only in mutant cells abutting the MF anteriorly, suggesting that accumulated CiFL in Nedd8 mutant cells is able to respond to Hh signaling (Ou, 2002).
CiFL accumulation in Nedd8 cells results from a defect in the machinery controlling CiFL protein processing. Ci protein processing is known to depend on the phosphorylation status of CiFL by PKA. The level of CiFL is downregulated when PKA is constitutively activated by the expression of its catalytic subunit. Therefore, the functional relationship between PKA activity and Nedd8 modification was examined. When the UAS-C* transgene was driven by eq-GAL4 for misexpression in the equator region of the eye disc, as visualized by the coexpressed GFP, the level of CiFL in the equator region was reduced, consistent with the observations that PKA phosphorylates Ci and promotes Ci proteolysis. Nedd8 mutant clones were then generated in the equator region where PKA is constitutively activated. In Nedd8 clones that overlap the eq-GAL4 expression domain, CiFL accumulates to a high level, identical to the level in the Nedd8 clone located externally to the eq-GAL4 expression domain. These results indicate that CiFL downregulation by PKA activity requires Nedd8 activity, and the effect of the Nedd8 pathway on CiFL processing is unlikely to be mediated through modulation of PKA activity (Ou, 2002).
The finding that CiFL accumulates in posterior smo3 clones indicates that Smo signaling contributes to the downregulation of CiFL in the posterior cells of the eye disc. This effect is in contrast to the smo role in the MF, where smo is required for CiFL activation. CiFL accumulation was also observed in the posterior Nedd8 mutant clones located proximally to the MF. In the smo3 Nedd8 double mutant clones, the level of CiFL is further enhanced, even in clones located distally to the MF, whereas no CiFL accumulation is detected in Nedd8 or smo3 clones, suggesting that Nedd8 and Smo function partially redundantly to downregulate Ci stability in the posterior cells of the eye disc (Ou, 2002).
The involvement of Nedd8 in controlling CiFL levels in the posterior cells of the eye disc suggests that Cullin proteins other than Cul1 may be involved in the posterior mechanism to control Ci stability. Among the mammalian Cullin family, Cul3 shares with the Cul1-based SCF complex the substrate CycE (Singer, 1999). To test whether Cul3 affects CiFL degradation in the eye disc, the available Drosophila Cul3 mutants were analyzed. CiFL accumulates in Cul3 mutant clones located posterior to the MF, with a higher level in nondifferentiating cells that surround differentiating photoreceptor clusters. In contrast, no CiFL accumulation is detected in anterior Cul3 mutant clones, indicating that Cul3 controls CiFL protein stability only in the posterior cells of the eye disc. Ci accumulation in posterior Cul3 mutant cells is controlled at the posttranscriptional level because ci expression is normal, as revealed by in situ hybridization. These results show that the CiFL degradation machinery in the posterior cells of the eye disc requires a Cul-3-mediated degradation mechanism. Ci accumulation is also detected in Cul3 mutant cells located in the A/P boundary of the wing disc. The level of Arm in Cul3 mutant clones in wing discs and the level of CycE in Cul3 mutant clones in eye discs remain constant, suggesting that Cul3 activity is specific to Ci (Ou, 2002).
In contrast to the Cul1-based SCFSlimb complex that controls CiFL processing only in the anterior cells of the eye disc, the Cul3-mediated Ci degradation mechanism is specific to the posterior cells. These specific activities in controlling Ci protein stability are not caused by differential gene expression of Cul1 and Cul3 in the eye disc. Ubiquitous mRNA expression patterns of both Cul1 and Cul3, and ubiquitous Cul1 protein expression are found all along the eye disc, suggesting that control of specificity is mediated by mechanisms other than regulation of Cul1 and Cul3 expression (Ou, 2002).
PKA phosphorylation promotes CiFL processing, and plays a role in the Hh signaling pathway for Ci activation. The requirement of PKA in CiFL degradation in the posterior cells of the eye disc was examined; CiFL downregulation is not regulated by PKA activity. Proteolytic processing of CiFL to the short form Ci75 is not a prerequisite for complete degradation in the posterior cells, in contrast to the proteolytic processing of the phosphorylated CiFL to the short form Ci75 in the anterior cells. To sum up, the results suggest that in the posterior cells of the eye disc, CiFL is degraded constitutively, and this degradation process is independent of PKA phosphorylation (Ou, 2002).
The COP9 signalosome (CSN) is an eight-subunit complex that regulates multiple signaling and cell cycle pathways. The CSN has been linked to the degradation of Cyclin E, which promotes the G1-S transition in the cell cycle and then is rapidly degraded by the ubiquitin-proteasome pathway. Using CSN4 and CSN5/Jab1 mutants, it has been shown that the CSN acts during Drosophila oogenesis to remove Nedd8 from Cullin1, a subunit of the SCF ubiquitin ligase. Overexpression of Cyclin E causes similar defects as mutations in CSN or SCFAgo subunits (see Archipelago) -- extra divisions or, in contrast, cell cycle arrest and polyploidy. Because the phenotypes are so similar and because CSN and Cyclin E mutations reciprocally suppress each other, Cyclin E appears to be the major target of the CSN during early oogenesis. Genetic interactions among CSN, SCF, and proteasome subunits further confirm CSN involvement in ubiquitin-mediated Cyclin E degradation (Doronkin, 2003).
The effect of the CSN on the activity of the SCF complex has been controversial. Although Nedd8 modification of Cullin1 stimulates SCF activity, the opposite process, deneddylation, has also been shown to be important for SCF function and cell cycle progression. For example, point mutations in the JAMM domain of the S. cerevisiae CSN5 homolog Rri abolish its deneddylation activity and enhance the growth defect shown by ts alleles of SCF genes. These results have led to the proposal that repeated cycles of neddylation and deneddylation are required for the sustained activity of the SCF. However, a recent gain-of-function analysis suggests that deneddylation by the CSN inhibits degradation of the SCF target p27kip1 (Doronkin, 2003).
The results of this study strongly support the idea that deneddylation of Cullin1 by the CSN is necessary for activity of the SCF complex. CSN mutations have the same, not opposite, effects on oogenesis as do Nedd8, cullin1, or ago mutations. CSN5 and CSN4 mutations also interact dominantly with cullin1 and ago mutations, further suggesting that the CSN works along with the SCF to promote Cyclin E degradation. These requirements for the CSN appear to demand its deneddylase activity, because the CSN5quo2 mutation, with a single amino acid substitution in the metalloprotease domain, behaves similarly to a CSN5 null (Doronkin, 2003).
Cycles of neddylation and deneddylation might control the association of an F box protein with an E3 ubiquitin ligase core complex or the association of a ubiquitin-loaded E2-conjugating enzyme with the E3 complex. Neddylation might also affect Cullin1 stability as suggested by the Cullin1 accumulation that is seen in CSN5 mutants and its reduction in Nedd8 mutants (Doronkin, 2003).
Conjugation of Nedd8 to cullins may regulate not only their activity, but also their subcellular distribution. Shuttling between the nucleus and cytoplasm has been proposed as a regulatory mechanism for E3 ubiquitin ligases when the target protein is ubiquitinated in the nucleus. The results showing that in CSN mutants, Nedd8-modified Cullin1 accumulates in the cytoplasm suggest that neddylation may be one way to regulate shuttling. Neddylation might favor nuclear export of Cullin1, and nuclear CSN would be required to remove Nedd8 and prevent export. Alternatively, neddylation might prevent Cullin1 nuclear import, and recycling of SCF into the nucleus would require cytoplasmic CSN. On either model, the CSN would be an important regulator of SCF activity. For example, modulation of SCF nuclear shuttling might affect the timing of Cyclin E degradation and entry into S phase of the cell cycle (Doronkin, 2003).
Mutations in Drosophila ago, the C. elegans gene cul1, or the F box-encoding lin23 have been shown to cause increased cell proliferation, suggesting a critical role for SCF in regulating cell divisions. Extra cell division is found to be a frequent phenotype produced by mutations in CSN5, CSN4, cullin1, ago, or by overexpression of Cyclin E. However, SCF and CSN mutations have also been shown to cause the opposite effect on the cell cycle. In null mutant clones of cullin1 or Nedd8, cell proliferation in Drosophila eye discs is arrested. Similarly, loss of CSN5, CSN4, Cullin1, or ago inhibits and finally stops cell proliferation and often leads to enlarged nuclei. The abundance of Cyclin E and giant polyploid nuclei are also present in mice that are mutant for cul1 (Doronkin, 2003).
Elevated levels of Cyclin E that may give cells a proliferative advantage are found in many human tumors. In many of these tumors the Cyclin E gene itself is amplified. However, among breast and ovarian cancer cell lines that overexpress Cyclin E protein without amplification, several lines have mutations in hCDC4, the human homolog of archipelago, suggesting that SCF[hcdc4] acts to suppress tumor formation. The results suggest that the CSN might have a similar effect (Doronkin, 2003).
In summary, these genetic and functional relationships between the CSN, the SCF, and the proteasome link these complexes in the regulation of Cyclin E degradation during normal development. When either the CSN or SCF are disrupted, the periodic degradation of Cyclin E is prevented, and cell cycle deregulation ensues (Doronkin, 2003).
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).
Cul1 is a core component of the evolutionarily conserved SCF-type ubiquitin ligases that target specific proteins for destruction. SCF action contributes to cell cycle progression but few of the key targets of its action have been identified. Expression of the mouse Cul1 (mCul1) in the larval wing disc of Drosophila has a dominant negative effect. It reduces, but does not eliminate, the function of SCF complexes, promotes accumulation of Cubitus interruptus (a target of SCF action), triggers apoptosis, and causes a small wing phenotype. A screen for mutations that dominantly modify this phenotype showed effective suppression upon reduction of E2F function, suggesting that compromised downregulation of E2F contributes to the phenotype. Partial inactivation of Cul1 delays the abrupt loss of E2F immunofluorescence beyond its normal point of downregulation at the onset of S phase. Additional screens showed that mild reduction in function of the F-box encoding gene slimb enhances the mCul1 overexpression phenotype. Cell cycle modulation of E2F levels is virtually absent in slimb mutant cells in which slimb function is severely reduced. This implicates Slimb, a known targeting subunit of SCF, in E2F downregulation. In addition, Slimb and E2F interacted in vitro in a phosphorylation-dependent manner. Thus the G1/S transcription factor E2F is an SCFSlmb target in Drosophila. These results argue that the SCFSlmb ubiquitin ligase directs E2F destruction in S phase (Hériché, 2003).
This study provides evidence indicating a role for an SCFSlmb complex in elimination of E2F during S phase in Drosophila. Mouse Cul1 overexpression has a dominant negative effect on SCF function in the wing imaginal disc resulting in apoptotic cell death leading to a small wing phenotype in adult flies. Thus, SCF activity normally suppresses cell death. The cell death that occurs upon reduction of Cul1 function is suppressed when E2F function is reduced by mutation. This genetic interaction might be due to action of E2F and Cul1 in parallel pathways, one suppressing and one enhancing cell death, or they might act in opposite directions in the same pathway. Because SCF complexes are involved in controlling protein degradation, the hypothesis is favored that Cul1 promotes the destruction of E2F and that the inappropriate persistence of E2F leads to cell death. Consistent with this idea, E2F overproduction can induce apoptosis. Furthermore, cell cycle specific destruction of E2F is suggested by the finding that E2F protein becomes undetectable in the synchronized S phase cells of the morphogenetic furrow of the eye imaginal disc. Double labelling analysis extended this finding to the asynchronously cycling cells of the wing imaginal disc and demonstrated that E2F is rapidly degraded prior to significant DNA replication. In contrast, it was found that E2F is detected in a fraction of S phase cells when SCF function is reduced by mCul1. This shows that normal E2F downregulation is delayed or slowed by reduction in SCF function (Hériché, 2003).
Although the dominant negative action of mCul1 in Drosophila was unanticipated, it offers a fortuitously convenient tool for the analysis of SCF function. The Drosophila Cul1 mutant is less useful because maternal contributions of Cul1 are so large that homozygous mutant animals develop to pupariation. Additionally, unlike the spatially restricted expression of mCul1, the strong Cul1 mutant alleles result in lethality. The stage and generality of the defects make analysis of the Cul1 mutant phenotype particularly difficult. In contrast, the induced expression of mCul1 in the wing disc does not compromise viability and it gives a distinctive graded phenotype well suited for the study of genetic modification. While the basis for the dominant negative effect of mCul1 remains unknown, it is important to recognize that inactivation of the endogenous Cul1 is incomplete so that a reduced level of Cul1 activity persists. One likely explanation for the negative effect is that mCul1 overexpression leads to sequestration of some limiting SCF components into weakly active complexes. This hypothesis is consistent with the fact that mCul1 retains some positive function in flies as revealed by its ability to prolong survival of dCul1 mutant flies (J.-K. Hériché and P.H. O'Farrell, unpublished data) (Hériché, 2003).
The essentially complete suppression of the mCul1 phenotype by reduction of the genetic dose of E2F suggests a remarkable level of specificity, in which, among the many targets of Cul1 action, the destruction of E2F appears to be particularly important. Similarly, limitations of Cul1 function in other organisms also uncovered the disproportionate importance of particular substrates. For example, Cul1 mutations in mice result in the accumulation of the SCF substrate cyclin E but not of p27, another well characterized SCF substrate. Among all of the substrates targeted for degradation by SCF in S. cerevisiae, it is the failure to degrade Sic1 that underlies the G1 arrest in cdc53 mutants. Thus, different substrates of the SCF appear to have particularly high dependence on SCF activity in different biological contexts. The experimental context using mCul1 expression in the wing disc is thought to be particularly effective in exposing the involvement of SCF in E2F destruction (Hériché, 2003).
Since SCF complexes function in conjunction with a variety of F-box proteins that act as specificity factors, mutations in individual F-box proteins ought to affect particular subsets of SCF substrates. In an extensive screen for loci that modify the reduction of function phenotype for Cul1, the gene encoding the F-box protein Slimb as a modifier was identified, but no contributions of other F-box encoding genes to the phenotype were detected. This implicates Slimb in the action of SCF on E2F. Analysis of E2F levels reveals that cell cycle oscillations in E2F levels were absent when Slimb function was severely reduced. Note that the severity of this E2F destruction phenotype in comparison to the mild defect in cell cycle programming of E2F destruction upon mCul1 expression is entirely consistent with the fact that the slimb mutant gives a stronger loss of function than the reduction of function imposed by mCul1 overexpression. The absence of cell cycle oscillation in E2F presence in the slimb mutant suggests that SCFSlmb is responsible for targeting E2F for S phase destruction. If this destruction is the consequence of direct action of SCFSlmb on E2F, the F-box protein would be expected to interact with E2F. This prediction was confirmed by pull-down experiments. Furthermore, it is demonstrated that the interaction between Slimb and E2F is dependent on phosphorylation, as expected for the interaction of F-box proteins with their substrates. The S phase specificity of E2F destruction is regulated by a targeting phosphorylation event, but this level of regulation has yet to be investigated (Hériché, 2003).
Since E2F is a positive regulator of the G1/S transition, it is not clear why S phase destruction of E2F is required. However, it is noted that slimb mutant cells do not replicate DNA normally and the replication defect is correlated with an increase in E2F in individual cells. This observation is consistent with observations indicating that E2F can limit DNA replication both in mammalian cells and in flies. This enigmatic feature of E2F regulation suggests that its continued presence has a negative effect on DNA replication and indicates that there is still much to learn about E2F roles in cell cycle regulation (Hériché, 2003).
SCFSlmb appears to influence other cell cycle events, perhaps by actions on other substrates, or perhaps as a result of events that are secondary to its influence on E2F destruction. For example, a slimb allele shows defects in centrosome duplication control, and slimb null cells undergo apoptosis. Both effects can be explained by a failure to inactivate E2F, since E2F promotes centrosome duplication in mammalian cells and E2F can induce apoptosis. A link between Slimb function and the RB/E2F pathway of cell proliferation control is also suggested by the genetic interaction between the C. elegans slimb ortholog lin-23 and the RBF ortholog lin-35 in which lin-35 function limits the severity of the loss of lin-23 . Since RBF is an important modifier of E2F activity, perhaps this interaction is a reflection of the action of both Lin-23 and Lin-35 on E2F. However, in the Drosophila system there was no modification of the mCul1 overexpression phenotype by mutations in either the RBF or DP gene, two known partners of E2F. This result suggests that these factors are not limiting factors in this situation or that E2F acts independently of RBF and DP. The latter hypothesis is the most likely explanation for the lack of an RBF interaction for the following reason. The E2F/RBF complex has to be disrupted for E2F to drive cells into S phase and E2F degradation occurs after the G1/S phase transition, which only happens after E2F has been released from its association with RBF. According to this view, it would be predicted that the mCul1-induced phenotype should be specifically sensitive to factors impinging on the elimination of E2F activity during S phase, and factors involved in the unknown processes by which persistence of E2F has negative outcome. Consequently, this experimental system may provide an avenue for the genetic dissection of this mysterious facet of E2F function (Hériché, 2003).
Thus, to explore potential roles for Cul1 in cell cycle control in D. melanogaster, mouse Cul1 (mCul1) was overexpressed in the wing imaginal disc. This overexpression has a dominant negative effect leading to a reduction in SCF function. The resulting small wing phenotype was used in a modifier screen to identify mutations in cell cycle genes capable of dominantly modifying the mCul1-induced phenotype. E2F loss-of-function mutations were the strongest modifiers since they completely suppress the phenotype. The reduction in SCF function associated with mCul1 overexpression also correlates with a failure to downregulate E2F normally in S phase. Additionally, mutations in slimb that reduce the function of the F-box protein Slimb enhance the phenotype and lead to persistence of E2F in S phase cells. Finally, this F-box protein interacts with E2F in vitro. These results indicate that an SCFSlmb complex is involved in regulating E2F activity during S phase (Hériché, 2003).
In Drosophila, egg development starts at the anterior tip of the ovary, in the germarium, where the germline stem cells divide to produce a cystoblast and a self-renewing stem cell. Each cystoblast undergoes four mitotic divisions with incomplete cytokinesis. The resulting 16 cells of each egg chamber are connected by intercellular bridges called ring canals. Exit from the cell cycle at the end of these four mitotic divisions requires the downregulation of Cyclin/Cdk activity. In the ovary of Drosophila, Encore activity is necessary in the germline to exit this division program (Ohlmeyer, 2003).
In encore mutant germaria, Cyclin A persists longer than in wild type. In addition, Cyclin E expression is not downregulated after the fourth mitosis and accumulates in a polyubiquitinated form. Mutations in genes coding for components of the ubiquitin-protease pathway such as cul1, UbcD2 and effete enhance the extra division phenotype of encore. Encore physically interacts with the proteasome, Cul1 and Cyclin E. The association of three factors, Cul1, phosphorylated Cyclin E, and the proteasome 19S-RP subunit S1, with the fusome is affected in encore mutant germaria. It is proposed that in encore mutant germaria the proteolysis machinery is less efficient and, in addition, reduced association of Cul1 and S1 with the fusome may compromise Cyclin E destruction and consequently promote an extra round of mitosis (Ohlmeyer, 2003).
Overexpression or loss-of-function mutations in a third group of genes such as Cyclin A, Cyclin B, Cyclin E and mutations in the gene encoding the E2 Ubiquitin conjugating enzyme UbcD1 lead to the production of cysts with 32 or 8 cells. These genes do not affect fusome integrity and thus timing and spatial characteristics of cell division appear to be intact. The encore gene belongs to this group of genes: its product is necessary for exit from mitosis. Loss of Encore activity results in egg chambers containing 32 rather than 16 cells. Mutations in the encore gene produce additional phenotypes, which show differential temperature sensitivity. encore mutant females raised at 18°C produce egg chambers with 16 cells, but they give rise to ventralized eggs. The extra cell division phenotype is only observed when encore mutant females are raised at high temperatures (25°-29°C). The encore gene encodes a 200 kDa protein with no homolog of a defined biochemical function. The mechanism by which Encore promotes exit from the cell cycle after four germline mitoses has been investigated (Ohlmeyer, 2003).
Cell cycle progression is controlled by a series of cell cycle dependent kinases (Cdk). Cdk activity is carefully regulated by the levels of the Cyclin subunits, by Cdk inhibitors (CKI) and by post-translational modification of the Cdk subunit through both activating and inactivating phosphorylation. Transition from G1 to S phase depends on Cdk2/Cyclin E activity, and on the timely destruction of the Cdk2/Cyclin E inhibitor p27. The Drosophila p27 homologue, Dacapo, is required for exit from the cell cycle in the embryo and eye imaginal disc. In addition, exit from the cell cycle requires destruction of the cyclins by the ubiquitin-proteasome system (UPS). The addition of ubiquitin requires three different activities; the ubiquitin activating enzyme (E1), the ubiquitin conjugating enzyme (E2) and the ubiquitin ligase enzyme (E3). The ubiquitinated protein bound to E3 is presented to the proteasome, isopeptidase activities in the 19S-recognition particle (RP) of the proteasome cleave the ubiquitin tail, the protein is unfolded and finally destroyed by the proteasome 20S-core particle (CP) (Ohlmeyer, 2003).
There are two E3 enzyme complexes that regulate the cell cycle progression. The first, the APC/cyclosome, regulates progression from G2 to M phase transition. The second, the SCF complex regulates the G1 to S phase transition. The SCF complex is composed of Skp/Cullin/Rbx1 and F-box proteins and controls substrate ubiquitination via an interaction between the F-box component and the phosphorylated target protein. In Drosophila and mammalian systems, mutations in the Cul3 and Ago genes have been shown to cause the accumulation of Cyclin E, entry to S-phase and doubling of cell number. Thus, proper regulation of the destruction machinery is important for maintaining normal levels of Cyclin E and assuring proper cell cycle progression (Ohlmeyer, 2003).
The work presented in this study demonstrates that the encore gene product associates with the SCF-ubiquitin-proteasome system and is required for proper exit from germline mitosis. The failure to downregulate Cyclin E after four cell divisions in conjunction with an accumulation of Cyclin A protein provide the conditions to promote an extra cell division. Encore can bind to Cul1, Cyclin E-Ub(n) and the proteasome. Cul1 and the proteasome 19S-RP subunit S1 are associated with the fusome and these associations are very much attenuated in encore mutant ovaries. It is proposed that as a direct consequence, Cyclin E is not degraded properly, its activity is misregulated and the cyst undergoes one extra cell division (Ohlmeyer, 2003).
This study shows that the fusome is a regulator of cell division during early oogenesis. Some of the functions ascribed to the fusome are to synchronize cyst mitosis and to provide the scaffold for the transport system necessary for oocyte determination. Limiting the number of cell divisions in the germarium could be achieved by regulating the association of proteins such as the cyclins and/or other cell cycle regulators with the fusome. The expression pattern of Cyclin A, Cul1, P-Cyclin E and 19S-S1 proteins in the germarium supports the idea that the fusome plays an important role in the regulation of mitosis. Indeed, Cyclin A association with the fusome is transient and occurs only during cyst division. In encore mutant germaria, Cyclin A remains associated with the fusome after cell division has stopped. Cul1 localization to the fusome suggests that the rest of the SCF complex also associates with the fusome and that substrate ubiquitination may happen at the fusome. The SCF component Cul1 is mainly associated with the fusome in the wild-type germaria. In encore mutant germaria, Cul1 localization to the fusome is very poor, leading to a proposal that this may be one reason why Cyclin E is not degraded properly. This also suggests that the degradation of Cyclin E and perhaps of other proteins degraded by the SCF-UPS may occur at the fusome. The association of P-Cyclin E supports this idea. The localization of P-Cyclin E in the wild type seems to be dynamic, consistent with the idea that the phosphorylated substrate is localized to the fusome, and then rapidly degraded via the SCF-UPS. In encore mutant germaria, the poor localization of Cul1 may result in an inefficient assembly of SCF complexes at the fusome. P-Cyclin E is localized to the fusome, but its degradation is compromised and as a result a consistent expression of P-Cyclin E is observed at the fusome. The partial association of the proteasome 19S-RP subunit S1 to the fusome supports the idea that proteolysis may occur at the fusome. The proteasome 19S-RP would recognize the polyubiquitinated substrate and recruit the rest of the proteasome to the fusome (Ohlmeyer, 2003).
The results suggest that Encore can associate with the SCF ubiquitin-proteasome system machinery and assists with the degradation of Cyclin E and perhaps other SCF substrates. Since the mutant Encore protein can still interact with SCF-UPS components, the mutant protein may form complexes but these might be inactive and/or the mutant protein poisons the degradation machinery. Consistent with such a hypothesis, the encore extra cell division phenotype is milder in hemizygous versus homozygous females at 25°C (Hawkins, 1996). Encore is required for the proper localization of Cul1, P-Cyclin E, S1 and presumably the rest of the proteolysis complex to the fusome. This localization may be more crucial at 29°C, whereas at lower temperatures a less efficient degradation system may have enough time for normal cell cycle regulation. encore mutations do not affect the 20S-Core Particle activity as measured by the rate of degradation of a fluorogenic peptide. It is not known whether Encore retains Cul1 at the fusome or whether Encore directly or indirectly modifies Cul1 in order to promote its localization at the fusome. Cul1 is known to be modified by the addition of Nedd8; however, Cul1 seems to be equally neddylated in encore and wild-type ovary extracts (Ohlmeyer, 2003).
In summary, the results suggest that the Encore protein assists with proper cell cycle progression in the Drosophila germarium by ensuring that Cul1 and the proteolysis machinery is localized at the mitosis coordination center, the fusome (Ohlmeyer, 2003).
Terminal differentiation of male germ cells in Drosophila and mammals requires extensive cytoarchitectural remodeling, the elimination of many organelles, and a large reduction in cell volume. The associated process, termed spermatid individualization, is facilitated by the apoptotic machinery, including caspases, but does not result in cell death. From a screen for genes defective in caspase activation in this system, a novel F-box protein, which was termed Nutcracker, was isolated that is strictly required for caspase activation and sperm differentiation. Nutcracker interacts through its F-box domain with members of a Cullin-1-based ubiquitin ligase complex (SCF): Cullin-1 and SkpA. This ubiquitin ligase does not regulate the stability of the caspase inhibitors DIAP1 and DIAP2, but physically binds Bruce, a BIR-containing giant protein involved in apoptosis regulation. Furthermore, nutcracker mutants disrupt proteasome activity without affecting their distribution. These findings define a new SCF complex required for caspase activation during sperm differentiation and highlight the role of regulated proteolysis during this process (Bader, 2010).
Most F-box proteins also possess another protein-interaction domain, usually comprising WD40 or LRR motifs, that is responsible for binding the ubiquitylation substrate. Nutcracker belongs to the class of F-box proteins that do not contain a known protein-protein interaction domain, and differs topologically from most F-box proteins in that its F-box domain is at the very C-terminus (Kirk, 2008). Sequence alignments with several F-box-only proteins revealed that Nutcracker shares some limited amino acid similarity with the mammalian FBXO7 protein, which also contains the F-box domain at the C-terminus. Although the sequence conservation is limited primarily to the F-box domain, it is possible that these two proteins share functional properties, as do other proteins that are conserved only within limited regions. For example, the C. elegans p53 protein displays less than 20% overall primary sequence similarity to the human protein, mostly in the active sites, but has been demonstrated to function in related cellular processes. Since FBXO7 has been shown to regulate the stability of cIAP1 (BIRC2) (Chang, 2006), it is possible that these two E3 ligases have a conserved function in caspase regulation (Bader, 2010).
The ubiquitin-proteasome system is implicated in regulating caspase activity. Several studies have shown that the ubiquitylation and degradation of DIAP1 is a means of displacing it from caspases when apoptosis is favored. Also, ubiquitylation of caspases themselves contributes to their regulation by preventing a critical mass of full-length caspases from auto-activation in a normal setting. The wide variety of other ubiquitin-modifying proteins that regulate apoptosis and caspase activity, including Bruce, Morgue and Uba1, imply the existence of an elaborate regulatory network that is controlled by ubiquitylation (Bader, 2010).
In the screen isolated another ubiquitin ligase, a Cullin-3-based complex, was isolated, indicating that caspase activation in this system is tightly controlled by ubiquitin modifications. These two complexes could regulate the stability of the same substrate, as is the case for regulation of Cubitus interruptus (Ci) stability in Hedgehog signaling by both Cullin-1-based and Cullin-3-based complexes, or they might target multiple important substrates. Alternatively, the E3 ligases isolated in the screen might play non-degradative roles in controlling caspase activity. For example, mono-ubiquitylation affects the targeted localization of proteins and these ubiquitin ligases might control the proper localization of caspase regulators. Another possibility is that these E3 ligases mediate the non-classical Lys63 ubiquitin chain addition that is important for protein-protein interaction. Thus, instead of degradation, these proteins might actually control interactions between caspase regulators (Bader, 2010).
Although DIAP1 is the only Drosophila BIR-containing protein that has been shown to directly inhibit caspases in vivo, Bruce has been implicated in modifying apoptosis in several death paradigms, and mutations in its mammalian homolog cause defects associated with excess cell death. This study showed that Bruce can physically bind Nutcracker, and that this interaction is independent of the F-box domain. Therefore, Bruce might be a substrate of Nutcracker. However, it was not possible to determine the steady-state levels of Bruce in nutcracker mutants, so it is as yet unclear whether it is indeed a substrate or a complex partner. The fact that Bruce also binds to another E3 ligase isolated in the screen suggests that this protein is a common regulator of caspase activation during individualization (Bader, 2010).
nutcracker mutants cause a reduction in proteasome activity. This decreased activity does not seem to be due to proteasome mislocalization or a reduction in their numbers, suggesting that Nutcracker controls proteasome activity directly. It is possible that Nutcracker modifies proteasome regulators, which could include, for example, proteins of the regulatory particle of the proteasome. An attractive model is that Nutcracker functions through proteasomes to activate caspases. Caspase activity is tightly controlled by the ubiquitin proteasome system. Therefore, it is possible that local activation of proteasomes controls localized caspase activation (Bader, 2010).
Many questions remain regarding the non-lethal role of caspases in cellular remodeling. For instance, is it a specialized activation that is governed by dedicated proteins, and to what extent are known apoptotic regulators involved in this process? Another intriguing question is how cells tolerate a certain level of caspase activation and avoid destruction by these potentially deadly proteases. Answers to these questions will not only uncover novel caspase regulators, but might also help us to understand how diseased cells, such as cancer cells, manage to escape cell death (Bader, 2010).
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