imaginal discs arrested: Biological Overview | References
Gene name - imaginal discs arrested
Cytological map position- 63F6-63F6
Function - signaling
Symbol - ida
FlyBase ID: FBgn0041147
Genetic map position - 3L:3,905,344..3,907,908 [+]
Classification - APC5 subunit of anaphase promoting complex
Cellular location - nuclear
The imaginal discs arrested (ida) gene that is required for proliferation of imaginal disc cells during Drosophila development. Ida is homologous to APC5, a subunit of the anaphase-promoting complex (APC/cyclosome). ida mRNA is detected in most cell types throughout development, but it accumulates to its highest levels during early embryogenesis. A maternal component of Ida is required for oogenesis and viable embryos. Homozygous ida mutants display mitotic defects: they die during prepupal development, lack all mature imaginal disc structures, and have abnormally small optic lobes. Cytological observations show that ida mutant brains have a high mitotic index and many imaginal cells contain an aneuploid number of aberrant overcondensed chromosomes. However, cells are not stalled in metaphase, since mitotic stages in which chromosomes are orientated at the equatorial plate are never observed. Interestingly, some APC/C-target substrates such as cyclin B are not degraded in ida mutants, whereas others controlling sister-chromatid separation appear to be turned over. Taken together, these results suggest a model in which IDA/APC5 controls regulatory subfunctions of the anaphase-promoting complex (Bentley, 2002; full text of article).
In holometabolous insects such as Drosophila, larval tissues are set aside from adult or imaginal tissues during early embryogenesis. During the instars, larval cells become polytenized and grow in size without cell division, and most will undergo a steroid-hormone-regulated programmed cell death at metamorphosis. By contrast, cells in the imaginal tissues proliferate and are maintained in a strictly diploid undifferentiated state throughout larval development. At metamorphosis, it is the imaginal cells that will differentiate to form the adult body plan. Because of these fundamental differences in cell fate, and because a maternal contribution is often sufficient to sustain all embryonic cell divisions, mutations in many genes regulating the diploid cell cycle do not cause lethality until metamorphosis. However, homozygous mutants eventually die because imaginal disc structures are not present to replace the histolyzing larval tissues. By examining mutants that display late-larval or prepupal lethal periods, many genes have been identified that play a role in regulating the diploid cell cycle. Thus, it is logical to predict that mutations in subunits of the anaphase-promoting complex would also display such phenotypes (Bentley, 2002).
The APC/cyclosome (APC/C) contains 8-13 subunits depending on species, and functions as an E3 ubiquitin-protein ligase that marks specific substrates for proteasome-dependent degradation. The APC/C generally controls the metaphase-to-anaphase transition and mitotic exit during cell division. Temporally regulated APC/C functions ensure that anaphase occurs only after successful DNA replication, proper microtubule attachment to all chromosomes via the kinetochore, and subsequent chromosome congression to the metaphase plate (reviewed by Skibbens, 1998; Page, 1999; Bentley, 2002 and references therein).
Much of the current understanding of the function of the APC/C is based primarily on biochemical and genetic work from fungal systems, including Saccharomyces cerevisiae, Schizosaccharomyces pombe and Aspergillus nidulans. Known functions of the APC/C complex include: degradation of the Securin or sister-chromatid separation inhibitor proteins, Pds1p and Cut2p; the regulated degradation of cyclins A, B and B3 during prometaphase, metaphase and anaphase; and continued B-type cyclin degradation through G1 until the onset of S phase. However, little is known about the biochemical function of individual APC/C subunits (Bentley, 2002 and references therein).
This study presents the cloning, characterization, and mutant analysis of the ida (imaginal discs arrested) gene. Mutations defining the ida gene have been mapped to the 63F3-7 region of chromosome 3 (Vaskova, 2000). ida encodes a homolog of the APC5 subunit (Yu, 1998) of the APC/C in Drosophila. The results are the first phenotypic characterization of APC5 mutations in metazoans. As expected, the data suggest that the APC5 subunit is required for late cell cycle events such as cyclin B degradation. However, based on cytological observations, an additional role for the APC5 subunit during chromosome congression is proposed, and a model is proposed in which IDA controls regulatory subfunctions of the APC/C (Bentley, 2002).
Animals null for the ida locus fail to undergo metamorphosis, stall in early prepupal development, and die 2-3 days later. Examination of ida third-instar larvae reveals that they are lacking normally developed excorporate and incorporate imaginal discs because diploid primordial cells fail to proliferate during larval development. Evidence is presented demonstrating that IDA is involved in germline cell proliferation, and a maternal component of ida+ is needed for egg production (Bentley, 2002).
A molecular characterization of ida shows that it encodes a protein with 23% identity and 61% similarity to APC5 from H. sapiens (Yu, 1998), a subunit of the APC/C. The APC/C is known to play essential roles in the ubiquitination of Securins and B-type cyclins. The degradation of these proteins is required for anaphase progression and exit from mitosis. Mutations in some APC/C subunits exist in S. cerevisiae and S. pombe that cause inviability and defects in cell cycle progression. These observations are consistent with the ida phenotype that describe in this study. The data support the theme that yeast and Drosophila share a functionally conserved set of APC/C proteins that regulate cell cycle progression and exit from mitosis (Bentley, 2002).
Because ida cells contain condensed chromosomes, they can enter mitosis. However, the high mitotic index seen in brain squashes suggests that cells have problems exiting mitosis. Both prometaphase and anaphase figures are frequently observed in ida mutants even though chromatin figures are severely hypercondensed. ida cells are aneuploid, and since the number of chromosomes is rarely a multiple of 8, this aneuploidy is probably a result of missegregated chromosomes during cell division. Thus at some level, ida cells are capable of progressing through the cell cycle (Bentley, 2002).
Only a subset of APC/C-dependent events is compromised in ida mutants. For example, in ida mutant cells attempting anaphase, cyclin B levels remain high. Consistent with these data, the hypercondensed chromosome figures in ida mutants are strikingly similar to those observed when a non-degradable form of cyclin B is overexpressed in proliferating cells. By contrast, other known APC/C-dependent events such as sister-chromatid separation can occur upon IDA depletion. This demonstrates that mutations in ida do not effect the APC/C-dependent degradation of Securin proteins. The fact that IDA is required for a fraction, but not all of the APC/C functions suggests that it does not play an essential role in the stability of the core complex, or in the ligation of ubiquitin oligomers to substrates (Bentley, 2002).
In Drosophila the APC/C complex is estimated to consist of 11 proteins. However the biochemical function and requirement of so many subunits is unclear. It is proposed that the large number of subunits reflects the need to identify and target a large number of substrates. The model is supported by the recent characterization of the 3D structure of the human APC/C. The structure has an asymmetric morphology with a large inner cavity surrounded by an outer protein wall. The complexity of the structure suggests that discrete subunits may guide substrates into the inner cavity, where ubiquitination could take place. Thus the removal of a single subunit would disrupt the ubiquitination of only a fraction of substrates. Interestingly, the data suggests that IDA may be involved in the degradation of cyclin B but is not essential for the degradation of Securins. It should be noted that in this model not all subunits need play a role in substrate identification, since some are required for core stability and catalyzing the ubiquitination events. For example, Cdc27 and Cdc16 play critical roles in core stability, and Apc11 is required for the ubiquitination of substrates (Bentley, 2002 and references therein).
Another consideration for the role of APC/C subunits concerns the possibility that they specifically interact with regulators of APC/C activity during the cell cycle. Perhaps the most actively studied regulators of APC/C activity are the components of the spindle checkpoint pathway. Upon detection of DNA damage or unattached kinetochores, the spindle checkpoint pathway will send a 'wait' signal. In response to this signal, Mad2 will bind the APC/C, preventing its activity and halt progression of all mitotic events until the checkpoint has been fulfilled. Positive regulators play an equally important role in driving the cell through coordinated mitotic events. In Drosophila, the WD40-repeat protein, Fizzy (FZY), binds to and drives APC/C-dependent ubiquitin-ligase activity in vitro. The FZY homolog in yeast, Cdc20p, positively regulates the destruction of Pds1p, and FZY is thought to serve a comparable role in Drosophila because FZY is required for Pimples (Securin) degradation during mitosis. Consistent with these predictions, loss-of-function mutations in fzy prohibit cells from progressing through metaphase, and demonstrate that FZY is required for metaphase exit and completion of mitosis in Drosophila. FZY is highly unstable and present only at late S phase and during mitosis, further ensuring that FZY-dependent APC/C events are temporally regulated. Finally, FZY degradation is dependent on APC/C subunits, demonstrating that FZY is also a substrate of the APC/C. An additional WD40-repeat protein, Fizzy-related (FZR), is also believed to be required for the degradation of B-type cyclins during M and G1 phases, but differs from FZY in that it is stable throughout the cell cycle (Bentley, 2002).
In ida mutants, cyclin B levels are not properly degraded during anaphase. Thus it is possible that the function of IDA, alone or in concert with other subunits, is to direct cyclin B to the APC/C for degradation. However, other defects are observed in ida cells that are distinct from those observed in cells expressing a non-degradable cyclin B transgene. Therefore, additional regulatory functions are proposed for the IDA protein to help explain the lack of metaphase figures, the observed sister-chromatid separation, the high levels of Bub1 staining during anaphase, and the resulting aneuploidy that is observed in ida mutant cells (Bentley, 2002).
In one model, IDA functions as a part of the APC/C that receives a spindle checkpoint 'wait' signal. Thus when IDA is missing, the spindle checkpoint signal is not received, but the cell initiates sister-chromatid separation and anaphase onset prematurely. Presumably, this could occur even in the absence of proper chromosome attachment and alignment at the metaphase plate. Thus, metaphase figures would not be observed in ida mutants, but aberrant anaphases containing lagging chromosomes with high Bub1 staining (equal to signal checkpoint firing) would be detected. The missegregation of the unattached chromatids would also lead to cells containing an aneuploid number of chromosomes (Bentley, 2002).
In an alternative model, IDA plays a role in targeting FZY for ubiquitin-dependent degradation. In this case, the removal of IDA would result in ectopic levels of FZY, which would prematurely activate sister-chromatid separation and progression through mitosis. Consistent with this model, mutations in ida suppress the embryonic lethality associated with a fizzy null mutation (Bentley, 2002).
It should be noted that neither model directly address the high mitotic index -- a hallmark of cell cycle stall -- observed in squashes of ida cells. However, it is proposed that as cells become more and more aneuploid, alternative pathways, including the DNA replication checkpoint, may eventually cause a prometaphase stall or arrest (Bentley, 2002).
Asymmetric cell divisions generate cell fate diversity during both invertebrate and vertebrate development. Drosophila neural progenitors or neuroblasts (NBs) each divide asymmetrically to produce a larger neuroblast and a smaller ganglion mother cell (GMC). The asymmetric localisation of neural cell fate determinants and their adapter proteins to the neuroblast cortex during mitosis facilitates their preferential segregation to the GMC upon cytokinesis. In this study a novel role is reported for the anaphase-promoting complex/cyclosome (APC/C) during this process. Attenuation of APC/C activity disrupts the asymmetric localisation of the adapter protein Miranda and its associated cargo proteins Staufen, Prospero and Brat, but not other components of the asymmetric division machinery. Miranda is ubiquitylated via its C-terminal domain; removal of this domain disrupts Miranda localisation and replacement of this domain with a ubiquitin moiety restores normal asymmetric Miranda localisation. These results demonstrate that APC/C activity and ubiquitylation of Miranda, in a proteasomal-independent process, are required for the asymmetric localisation of Miranda and its cargo proteins to the NB cortex (Slack, 2007).
A recently published study reported the isolated a novel allele of imaginal discs arrested (ida), homozygotes of which survive until early pupal stages of development and fail to properly localise Miranda to the basal cortex of mitotic neuroblasts (Slack, 2006). Sequence analysis of this allele, idaPL17, revealed a single nucleotide transversion within the ida coding sequence, resulting in a premature stop codon at aa 334 (Q334-->stop), the first residue of a putative tetratricopeptide (TPR) motif. In mutant larvae, only 58% of prophase NBs properly localised Miranda to the cortex compared with 100% of wild-type prophase NBs. A total of 41% of idaPL17 metaphase NBs showed Miranda accumulation in a pericentrosomal compartment at the expense of cortically localised protein, with 8% of mutant NBs showing a complete loss of cortically localised protein, whereas 91% of wild-type metaphase NBs showed cortically localised protein only. At low frequency, anaphase cells were observed, as defined by separated chromosome populations displaced towards opposite poles of the cell, confirming previous observations (Bentley, 2002) that ida mutant neuroblasts are not arrested at metaphase. Miranda was still mislocalised to pericentrosomal regions in these anaphase neuroblasts. No phenotypic defects were detected in idaPL17 homozygous mutant embryos, presumably due to the perdurance of maternally provided protein, and NB clones induced at early larval stages did not show any obvious mitotic or Miranda-localisation defects, again indicative of protein stability. Attempts to induce maternal germline clones homozygous for idaPL17 did not yield any surviving embryos, suggesting an essential requirement for ida function during oogenesis. The idaPL17 mutant appears to be a genetic null, because the penetrance of the Miranda localisation phenotype did not increase in hemizygotes over a small deficiency that removes the entire ida locus or in transheterozygotes over the mRNA null allele, idaD14. The defects in Miranda localisation in ida mutant NBs could not be rescued by expressing a GFP::Ida fusion protein, which, at all stages of the cell cycle, was localised throughout the cytoplasm (Slack, 2007).
ida encodes the Drosophila homologue of human APC5, a subunit of the APC/C multiprotein complex (Bentley, 2002). In order to determine whether the defects in Miranda localisation are a specific consequence of ida loss of function or are caused by a more general disruption to APC/C activity, the effect was analyzed of loss-of-function mutations in genes encoding other APC/C subunits. A significant number of mitotic NBs with pericentrosomal Miranda accumulation was observed in animals homozygous for mutations in either cdc27 or morula (APC2), suggesting that Miranda asymmetric cortical localisation is disrupted when APC/C activity is attenuated. However, no defects in Miranda localisation were observed in homozygous mutants for strong loss-of-function alleles for the two APC/C activators, cdc20 (fizzy) or cdh1 (retina aberrant in pattern/fizzy related), indicating that Miranda targeting might occur independently of these two proteins. Loss of ida function causes several mitotic defects, including an increased mitotic index, loss of cyclin B degradation and hypercondensed chromosomes (Bentley, 2002). However, a strong hypomorphic allele of polo that shows similar mitotic defects had normal cortical Miranda localisation. Furthermore, colchicine treatment of wild-type NBs to depolymerise microtubules and induce metaphase arrest did not disrupt Miranda localisation, therefore suggesting that the ida mutant phenotype is not a secondary consequence of a delay or block in mitosis (Slack, 2007).
In C. elegans, APC/C function during embryonic anteroposterior axis formation promotes the association of the paternal pronucleus/centrosome with the embryonic cortex (Rappleye, 2002). The pericentrosomal accumulation of Miranda in APC/C mutant NBs led to an investigation of a possible requirement for centrosomal function during Miranda localisation. Loss-of-function mutants for centrosomin (cnn), which encodes a core component of the centrosome in Drosophila and is required for proper centrosome assembly, were examined and found that Miranda localisation to the NB cortex was normal in these mutants. Furthermore, cnn; ida double mutants still accumulated pericentrosomal Miranda, suggesting that the ida mutant phenotype is not dependent on intact centrosomal function. It was also noted that complete colocalisation of Miranda with Centrosomin was not seen in ida mutant NBs, suggesting that Miranda is localised in a separate compartment that itself localises to a region near to the centrosome. Accumulation of Miranda in ida mutants was insensitive to colchicine treatment to depolymerise microtubules, suggesting that the ida mutant phenotype is not dependent on the mitotic spindle, although the compartment in which Miranda localises separates from the region of the centrosome upon colchicine treatment. Pericentrosomal accumulation of Miranda was also observed in ida mutants after latrunculin treatment, suggesting that the ida mutant phenotype occurs independently of intact actin filaments (Slack, 2007).
So far this pericentrosomal compartment has not been identified, although colocalisation was not observed with either Rab11 or Nuclear fallout (Nuf), both of which are markers for recycling endosomes, suggesting that pericentrosomal Miranda accumulation occurs independently of the recycling endosomal machinery. Miranda has been shown to localise to the centrosome both in biochemical and immunohistochemical studies, but, under the conditions used for the current experiments, significant levels of centrosomal Miranda localisation was not observed in wild-type NBs (Slack, 2007).
The localisation of Miranda to the NB basal cortex requires the correct localisation of the apical protein complex, which includes Inscuteable, Pins and aPKC. In ida mutant NBs, Inscuteable localises normally to the apical cortex during early prophase and is maintained as an apical cortical crescent during metaphase. Similar results were obtained using anti-aPKC and anti-Pins, suggesting that the defects in Miranda localisation in ida mutant NBs is not caused by a disruption to the apical complex and that the APC/C functions downstream of or in parallel to the apical components (Slack, 2007).
Miranda acts as an adapter protein for the cell fate determinant Prospero, for the prospero mRNA adapter Staufen and for the translational repressor Brat. In ida mutant NBs, Prospero, Staufen and Brat all lose their cortical localisation and colocalise with Miranda pericentrosomally, suggesting that they are still able to complex with Miranda. By contrast, the cortical localisation of both Numb and PON was unaffected by loss of ida function, and both Numb and PON formed normal cortical crescents in ida mutant NBs that showed strong Miranda mislocalisation defects, suggesting that ida function is required specifically for the localisation of Miranda and its associated cargo proteins to the NB basal cortex (Slack, 2007).
The ida mutant phenotype resembles that seen in mutants for the tumour suppressor lgl, in which the targeting of Miranda and other basally localised molecules to the NB cortex is disrupted so that Miranda no longer forms a cortical crescent at metaphase but is instead mislocalised to the centrosomes and mitotic spindle. It was reasoned that, if the ida mutant phenotype resulted from a reduction of Lgl function, then further reducing Lgl activity should enhance the Miranda mislocalisation phenotype. However, removing one copy of lgl using a null allele had no effect on the ida mutant phenotype and the number of NBs with pericentrosomal Miranda was comparable to mutants for ida alone (49% of NBs with pericentrosomal Miranda), suggesting that Lgl is not a downstream effector of Ida activity. By contrast, removing one copy of miranda using a deficiency that removes the entire miranda locus [Df(3R)ora19] in the idaPL17 mutant background strongly suppressed the Miranda mislocalisation phenotype, suggesting that Miranda itself might be a target for APC/C activity (Slack, 2007).
These data demonstrate that the correct localisation of Miranda to the basal NB cortex requires APC/C. Because the APC/C normally functions as an E3 ubiquitin ligase, whether this role of the APC/C could be mediating the effects on Miranda localisation was examined. In order to determine whether Miranda can be ubiquitylated, immunoprecipitations were performed on protein extracts of Drosophila S2 cells in which FLAG-tagged Miranda and expressed HA-tagged ubiquitin were constitutively expressed under the control of a heat-shock promoter. After immunoprecipitation using anti-HA antibody, FLAG-Miranda was detected in the immune complex in extracts only from cells in which HA-ubiquitin expression had been induced by heat shock. Immunoprecipitations were performed on protein extracts both from S2 cells and larval brains expressing only FLAG-Miranda without expressing exogenous HA-tagged ubiquitin. In both cases, ubiquitylated Miranda was detected in the immune complex after immunoprecipitation using anti-FLAG antibodies. These results using brain and S2 extracts clearly demonstrate that Miranda can be ubiquitylated both in vivo and in S2 cells. Although the antibody used recognises both mono- and poly-ubiquitin conjugates, only a single band was observed on the western blots probed for ubiquitylated Miranda. The absence of higher molecular weight Miranda species, even in the presence of proteasome inhibitors, suggests that Miranda might be mono- rather than poly-ubiquitylated (Slack, 2007).
The C-terminal region of Miranda contains a putative APC/C-recognition motif (amino acids 811 to 814: GKEN) that shows homology to the KEN box, a motif required for Cdh1-dependent APC/C-mediated ubiquitylation of substrate proteins (Castro, 2003). To examine the effects of the removal of this motif on Miranda localisation, the mirandaRR127 allele was used; in this allele the C-terminal 103 amino acids of the encoded protein, including the GKEN motif, are replaced by an unrelated stretch of 112 amino acids. The localisation of this truncated form of Miranda was examined in mitotic larval NBs by generating somatic clones using the MARCM system, which allows the generation of homozygous-mutant NB clones that express membrane-bound CD8::GFP in an otherwise heterozygous background. In mirandaRR127 mutant NBs, Miranda localisation was similar to that seen in ida mutant NBs: the truncated protein was exclusively cytoplasmic during early prophase and accumulated in a pericentrosomal compartment at the expense of cortical protein during metaphase. As in ida mutant NBs, accumulation of Miranda in mirandaRR127 mutant NBs was insensitive to colchicine treatment to depolymerise microtubules, and accumulation of pericentrosomal Prospero was also observed in these mutant NBs. Removal of this C-terminal domain prevented ubiquitylation of Miranda in S2 cells. Furthermore, NBs that overexpress this truncated protein showed a similar mislocalisation of the expressed protein and replacement of this C-terminal domain with ubiquitin restored normal localisation. Interestingly, mutation of the GKEN motif itself did not prevent Miranda ubiquitylation and had no effect on the localisation of the protein, suggesting that mutation of this site alone is insufficient to disrupt ubiquitylation of Miranda (Slack, 2007).
A high proportion of embryonic mirandaRR127 mutant NBs was observed in which Miranda was localised pericentrosomally at the expense of cortical protein. The observations that both embryonic and larval NBs mutant for the mirandaRR127 allele showed some cortical localisation although at a much reduced level and the presence of low levels of cortical Miranda in embryos derived from mirandaRR127 germline clones suggest that the mutant protein produced by the mirandaRR127 allele retains some ability to localise to the cortex. Ubiquitylation is clearly an important aspect of the Miranda localisation process, but in its absence a proportion of the Miranda present in the NB can be localised at the basal cortex by other mechanisms. Hence, the inability to ubiquitylate Miranda in the mirandaRR127 mutant causes only an incomplete loss of localisation (Slack, 2007).
Ubiquitylation by the APC/C normally targets proteins for degradation via the 26S proteasome. Although it is possible that a proportion of ubiquitylated Miranda is targeted for degradation, disruption to proteasome function caused markedly different phenotypes than those observed when APC/C activity was attenuated. Although Miranda was observed accumulating in the region of the centrosomes in NBs mutant for the proteasome regulatory subunits Rpn6 or Tbp-1, this process was microtubule dependent, whereas in both ida mutant NBs and NBs mutant for the mirandaRR127 allele, accumulation of Miranda was observed even after microtubule depolymerisation with colchicine. In addition, pericentrosomal accumulation of either Prospero or Staufen was not seen in proteasome-mutant NBs. Furthermore, no significant differences by western blot were observed in Miranda protein levels between wild-type and ida mutant brain extracts, suggesting that the pericentrosomal localisation of Miranda in ida mutants is not caused by excessive Miranda accumulation and therefore reflects the disruption of a process other than proteasomal degradation. Several proteasome-independent processes regulated by ubiquitylation have been identified, including protein kinase activation, vesicle trafficking, DNA repair and gene silencing (Slack, 2007).
The asymmetric localisation of cell fate determinants during NB division is tightly coordinated with changes in the cell cycle. The formation of an apical complex of proteins during early prophase not only directs the correct orientation of the mitotic spindle during metaphase but is also required for the formation of a basal crescent of cell fate determinants and their adapter molecules during late prophase/metaphase. It thus appears likely that multiple components of the cell cycle machinery that coordinate cell cycle transitions might also be involved in the regulation of basal protein localisation, as has been shown for cdc2. This study has shown that the efficient localisation of the adapter protein Miranda to the NB basal cortex requires the activity of the APC/C mitotic regulator. Mutations in several APC/C core subunits showed reduced cortically localised Miranda, with cytosolic accumulation of Miranda in an as yet unidentified pericentrosomal compartment. By contrast, apical complex formation was unaffected in these mutant NBs, showing that the APC/C acts downstream of or in parallel to the apical complex to ensure proper basal protein localisation. Furthermore, the basal localisation of PON/Numb were also unaffected by loss of APC/C activity, suggesting that Miranda itself might be a specific target for APC/C activity in mitotic NBs. This is further supported by the observation that the ida mutant phenotype can be partially rescued by specifically reducing Miranda protein levels (Slack, 2007).
The APC/C functions as an E3 ubiquitin ligase, and this study has shown that Miranda is a ubiquitylated protein in both cultured cells and larval NBs. Extensive attempts to demonstrate that Miranda ubiquitylation is APC/C-dependent have proved inconclusive. Therefore, the possibility cannot be ruled out that the effects of loss of APC/C activity on Miranda localisation might be indirect. However, the phenotypes observed in APC/C mutants are recapitulated in the mirandaRR127 allele encoding a C-terminal truncation of the protein. Not only are both Miranda localisation and ubiquitylation dependent on this region of protein, but replacement of this domain with ubiquitin is able to restore wild-type protein localisation. Although the C-terminal domain of Miranda is clearly required for ubiquitylation and cortical localisation of the protein, the precise ubiquitylation sites in Miranda are as yet unknown and will be an interesting area for further study. It is therefore speculated that the pericentrosomal accumulation of Miranda and the reduction of basally localised protein in APC/C mutants might reflect a loss of Miranda ubiquitylation. Recently, it has been shown that the ubiquitin moiety itself can function as a protein-protein interaction domain. Ubiquitylation of Miranda could function as a signal to regulate its transport to the basal cell cortex perhaps by influencing its association with motor proteins that mediate basal protein targeting. Alternatively, ubiquitylation of Miranda could regulate the retention of Miranda at the basal cell cortex by influencing its association with anchoring or scaffolding molecules. Efficient localisation and/or retention of Miranda to the basal cortex clearly requires APC/C activity, but the presence of Miranda protein at the basal cortex in APC/C mutants, albeit at a much reduced level compared with wild-type, indicates that other processes and molecules might also be involved (Slack, 2007).
The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit ubiquitin ligase that mediates the proteolysis of cell cycle proteins in mitosis and G(1). A yeast three-hybrid screen was used to identify proteins that interact with the internal ribosome entry site (IRES) of mammalian platelet-derived growth factor 2 mRNA. Surprisingly, this screen identified Apc5, although it does not harbor a classical RNA binding domain. It was found that Apc5 binds the poly(A) binding protein (PABP), which directly binds the IRES element. PABP was found to enhance IRES-mediated translation, whereas Apc5 overexpression counteracted this effect. In addition to its association with the APC/C complex, Apc5 binds much heavier complexes and cosediments with the ribosomal fraction. In contrast to Apc3, which is associated only with the APC/C and remains intact during differentiation, Apc5 is degraded upon megakaryocytic differentiation in correlation with IRES activation. Expression of Apc5 in differentiated cells abolished IRES activation. This is the first report implying an additional role for an APC/C subunit, apart from its being part of the APC/C complex (Koloteva-Levine, 2004; full text of article).
Unlike viral internal ribosome entry sites (IRESs), which are usually robust and efficient, the cellular IRESs are often weak and their requirements for IRES trans-acting factors (ITAFs) are more stringent. This allows the cellular IRES elements to serve as modulators of translation in response to specific physiological signals. An active IRES musters the translation machinery to secondary/tertiary structures within the 5' untranslated region (UTR) upstream of the initiator AUG codon. The proteins regulating this recruitment are largely unknown. Attempts were made to identify these ITAFs by using a three-hybrid screen that selects for RNA-protein interactions in the nucleus of a living yeast cell by supporting the formation of a stable complex with a specific RNA bait. A human HeLa cell cDNA expression library and a portion of the IRES element of human platelet-derived growth factor 2 (PDGF2) mRNA were used as the RNA bait. One of the cDNA fragments selected by the screen turned out to encode the 361-amino-acid-long carboxy-terminal part of the 755-amino-acid-long human Apc5 protein (Koloteva-Levine, 2004).
The anaphase-promoting complex/cyclosome (APC/C) is the ubiquitin ligase complex that mediates the degradation of at least 15 different proteins during mitosis and G1. APC/C degradation substrates include mitotic kinases such as cyclins A and B, plk1, aurora and nek kinases, the metaphase inhibitor securin, the Xkid motor protein, cdc6 and geminin (which are involved in the regulation of DNA synthesis), the product of the APC/C regulator gene fzy, and several other proteins. The mammalian APC/C comprises 12 subunits and is regulated by interaction with additional proteins and by phosphorylation of several of its subunits. The large size and complex structure of the APC/C are puzzling, especially in view of the fact that the Apc11 subunit is capable of mediating ubiquitination on its own. It is possible that several of its subunits act to connect the APC/C with regulatory pathways that control cell cycle progression under normal conditions and in response to signals, albeit the specific function of most of the subunits is unknown. There has been speculation regarding the role of some of the subunits (e.g., Apc1, Apc2, Apc3, Apc6, and Apc11), based on weak homologies to other proteins. More specifically, the Apc2 and Apc11 subunits are related to components of SCF, another ubiquitin ligase complex, Apc1 shares a structural motif with the two large subunits of the 19S cap complex of the 26S proteasome. Apc5 does not have sequence similarity to any protein of known function (Koloteva-Levine, 2004).
Although the Apc5 protein does not contain any classical RNA-binding motif, it was tempting to pursue the possible functional connection between this cell cycle-related protein and IRES function. This was particularly intriguing in view of the fact that the G2/M cell cycle phase was shown to be specifically permissive to the function of several IRES elements, such as those of hepatitis C virus, ODC and c-myc, and the p58PITSLRE cyclin-dependent kinase. Moreover, a certain time window during the course of differentiation also proved to be specifically more permissive for IRES function. Apc5 is essential to viability in budding yeast and in Drosophila, but its function is not clear. The human Apc5 was studied in logarithmically growing and differentiated human K562 cells and verified its effect on IRES-mediated translation (Koloteva-Levine, 2004.
This study shows that Apc5 is degraded during megakaryocytic differentiation, in correlation with IRES activation. Apc5 overexpression inhibits differentiation-induced IRES activation. Apart from the primary nuclear location of Apc5 and its association with the APC/C, it is also associated with heavier complexes and with the ribosomal salt wash (RSW) fraction. This is in contrast to Apc3, which is tightly bound to the APC/C, does not take part in other complexes, and is maintained at a constant level during differentiation. In addition, Apc5 binds to poly(A) binding protein (PABP), PABP enhances PDGF2 IRES activity, and Apc5 interferes with PABP-related IRES activation. This is the first report implying an additional role for an APC/C subunit, apart from forming part of the APC/C E3 complex (Koloteva-Levine, 2004).
The ubiquitin-targeting pathway is evolutionarily conserved and critical for many cellular functions. A role has been discovered for two ubiquitin-protein ligases (E3s), Rsp5p and the Apc5p subunit of the anaphase-promoting complex (APC), in mitotic chromatin assembly in Saccharomyces cerevisiae (Harkness, 2002). The present study investigated whether Rsp5p and Apc5p interact in an intracellular pathway regulating chromatin remodeling. Genetic studies strongly suggest that Rsp5p and Apc5p do interact and that Rsp5p acts upstream of Apc5p. Since E3 enzymes typically require the action of a ubiquitin-conjugating enzyme (E2), E2 mutants were screened for chromatin assembly defects; this screen resulted in the identification of Cdc34p and Ubc7p. Cdc34p is the E2 component of the SCF (Skp1p/Cdc53p/F-box protein). Therefore, additional SCF mutants were analzyed for chromatin assembly defects. Defective chromatin assembly extracts generated from strains harboring a mutation in the Cdc53p SCF subunit or a nondegradable SCF target, Sic1δphos, confirmed that the SCF was involved in mitotic chromatin assembly. Furthermore, Ubc7p was shown to physically and genetically interacts with Rsp5p, suggesting that Ubc7p acts as an E2 for Rsp5p. However, rsp5CA and δubc7 mutations had opposite genetic effects on apc5CA and cdc34-2 phenotypes. Therefore, the antagonistic interplay between δubc7 and rsp5CA, with respect to cdc34-2 and apc5CA, indicates that the outcome of Rsp5p's interaction with Cdc34p and Apc5p may depend on the E2 interacting with Rsp5p (Arnason, 2005).
The observations presented in this report provide insight into the breadth of the regulatory network defined by the ubiquitin-protein ligases Apc5p and Rsp5p. The position of Rsp5p on the cytosolic face of the plasma membrane places it in an ideal location to receive extracellular signals. Rsp5p can then respond to these signals by setting a signaling cascade in motion. Glucose signaling antagonistically controls the APC (inhibits) and the SCF (activates). Thus, Rsp5p could, in part, mediate the response of the APC and the SCF to glucose, as glucose triggers the Rsp5p-dependent turnover of some proteins. The complex phenotypic spectrum exhibited by rsp5 mutants could be explained by the potentially elaborate interaction of Rsp5p with multiple E2s. This study shows that the interaction of Rsp5p with Ubc7p may be specific for mitotic chromatin assembly. Finally, this study raises many interesting and exciting questions. For example, what is the nature of the Rsp5p-SCF interaction? Furthermore, how does the signal received by Rsp5p reach the APC? This question invokes the involvement of a protein capable of shuttling across the nuclear membrane (Arnason, 2005).
The anaphase-promoting complex/cyclosome (APC/C) is a multicomponent E3 ubiquitin ligase that, by targeting protein substrates for 26S proteasome-mediated degradation through ubiquitination, coordinates the temporal progression of eukaryotic cells through mitosis and the subsequent G1 phase of the cell cycle. Other functions of the APC/C are, however, less well defined. This study shows that two APC/C components, APC5 and APC7, interact directly with the coactivators CBP and p300 through protein-protein interaction domains that are evolutionarily conserved in adenovirus E1A. This interaction stimulates intrinsic CBP/p300 acetyltransferase activity and potentiates CBP/p300-dependent transcription. APC5 and APC7 suppress E1A-mediated transformation in a CBP/p300-dependent manner, indicating that these components of the APC/C may be targeted during cellular transformation. Furthermore, CBP is required in APC/C function; specifically, gene ablation of CBP by RNA-mediated interference markedly reduces the E3 ubiquitin ligase activity of the APC/C and the progression of cells through mitosis. Taken together, these results define discrete roles for the APC/C-CBP/p300 complexes in growth regulation (Turnell, 2005).
Arnason, T. G., Pisclevich, M. G., Dash, M. D., Davies, G. F. and Harkness, T. A. A. (2005). Novel interaction between Apc5p and Rsp5p in an intracellular signaling pathway in Saccharomyces cerevisiae. Eukaryot Cell 4: 134-146. PubMed ID: 15643069
Bentley, A. M., Williams, B. C., Goldberg, M. L. and Andres, A. J. (2002). Phenotypic characterization of Drosophila ida mutants: defining the role of APC5 in cell cycle progression. J. Cell Sci. 115: 949-961. PubMed ID: 11870214
Castro, A., Vigneron, S., Bernis, C., Labbe, J. C. and Lorca, T. (2003). Xkid is degraded in a D-box, KEN-box, and A-box-independent pathway. Mol. Cell. Biol. 23: 4126-4138. PubMed ID: 12773557
Harkness, T. A. A., Davies, G. F. Ramaswamy, V. and Arnason, T. G. (2002). The ubiquitin-dependent targeting pathway in Saccharomyces cerevisiae plays a critical role in multiple chromatin assembly regulatory steps. Genetics 162: 615-632. PubMed ID: 12399376
Koloteva-Levine, N., et al. (2004). The Apc5 subunit of the anaphase-promoting complex/cyclosome interacts with poly(A) binding protein and represses internal ribosome entry site-mediated translation. Mol. Cell. Biol. 24(9): 3577-87. PubMed ID: 15082755
Page, A. M. and Hieter, P. (1999). The anaphase-promoting complex: new subunits and regulators. Annu. Rev. Biochem. 68: 583-609. PubMed ID: 10872461
Rappleye, C. A., Tagawa, A., Lyczak, R., Bowerman, B. and Aroian, R. V. (2002). The anaphase-promoting complex and separin are required for embryonic anterior-posterior axis formation. Dev. Cell 2: 195-206. PubMed ID: 11832245
Skibbens, R. V. and Hieter, P. (1998). Kinetochores and the checkpoint mechanism that monitors for defects in the chromosome segregation machinery. Annu. Rev. Genet. 32: 307-337. PubMed ID: 9928483
Slack, C., Somers, W., Sousa-Nunes, R., Chia, W. and Overton, P. (2006). A mosaic genetic screen for novel mutations affecting Drosophila neuroblast divisions. BMC Genet. 7: 33. PubMed ID: 16749923
Slack, C., Overton, P. M., Tuxworth, R. I. and Chia, W. (2007). Asymmetric localisation of Miranda and its cargo proteins during neuroblast division requires the anaphase-promoting complex/cyclosome. Development 134(21): 3781-7. PubMed ID: 17933789
Turnell, A. S., et al. (2005). The APC/C and CBP/p300 cooperate to regulate transcription and cell-cycle progression. Nature 438(7068): 690-5. PubMed ID: 16319895
Vaskova, M., Bentley, A. M., Marshall, S., Reid, P., Thummel, C. S. and Andres, A. J. (2000). Genetic analysis of the Drosophila 63F early puff. Characterization of mutations in E63-1 and maggie, a putative Tom22. Genetics 156: 229-244. PubMed ID: 10978288
Yu, H., Peters, J. M., King, R. W., Page, A. M., Hieter, P. and Kirschner, M. W. (1998). Identification of a cullin homology region in a subunit of the anaphase-promoting complex. Science 279: 1219-1222. PubMed ID: 9469815
date revised: 27 January 2008
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