Cdc27: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - Cell division cycle 27

Synonyms - mákos (mks)

Cytological map position - 65E9

Function - signaling

Keywords - cell cycle, anaphase-promoting complex (APC), protein degradation

Symbol Symbol - Cdc27

FlyBase ID: FBgn0012058

Genetic map position - 2L

Classification - Tetratricopeptide repeat protein

Cellular location - cytoplasmic

NCBI links: Entrez Gene
Cdc27 orthologs: Biolitmine

Progression through mitosis requires the ubiquitin-mediated proteolysis of several regulatory proteins. A large multisubunit complex known as the anaphase-promoting complex or cyclosome (APC/C) plays a key role as an E3 ubiquitin-protein ligase in this process. The APC/C adds chains of ubiquitin to substrate proteins, targeting them for proteolysis by the 26S proteasome. The gene makos (mks) encodes the Drosophila counterpart of the Cdc27 subunit of the anaphase promoting complex (APC/C). Neuroblasts from third-larval-instar mks mutants arrest mitosis in a metaphase-like state but show some separation of sister chromatids. In contrast to metaphase-checkpoint-arrested cells, such mutant neuroblasts contain elevated levels not only of cyclin B but also of cyclin A. Mutations in mks enhance the reduced ability of hypomorphic polo mutant alleles to recruit and/or maintain the centrosomal antigens gamma-tubulin and CP190 at the spindle poles. Absence of the MPM2 epitope from the spindle poles in such double mutants suggests Polo kinase is not fully activated at this location. Thus, it appears that spindle pole functions of Polo kinase require the degradation of early mitotic targets of the APC/C, such as cyclin A, or other specific proteins. The metaphase-like arrest of mks mutants cannot be overcome by mutations in the spindle integrity checkpoint gene bub1, confirming this surveillance pathway has to operate through the APC/C. However, mutations in the twins/aar gene, which encodes the 55kDa regulatory subunit of PP2A, do suppress the mks metaphase arrest and so permit an alternative means of initiating anaphase. Thus the APC/C might normally be required to inactivate wild-type twins/aar gene product (Deak, 2003).

Substrates of the APC/C include the mitotic cyclins A, B and B3, which are degraded during prometaphase and the early and late stages of anaphase, respectively. Separation of sister chromatids also depends upon APC/C-mediated proteolysis of the Securin inhibitor proteins Pds1 and Cut2 in the budding and fission yeasts, respectively and their counterpart Pimples in Drosophila (Deak, 2003 and references therein).

There have been few studies of the roles of individual APC/C components. The core catalytic activity of the APC/C appears to reside with the Zn2+-chelating APC11 subunit, which appears able to provide ubiquitin ligase activity alone in vitro in conjunction with Ubc4 as an E2 ubiquitin transfer enzyme. However, in order to achieve ubiquitination in vitro using UbcH10 as an E2 enzyme, APC11 has to be partnered by APC2 (Deak, 2003 and references therein).

In Drosophila, studies directed at the genes for four APC/C components have suggested different functions. The APC2 subunit, Morula, is required not only to mediate progression through metaphase in mitotically cycling cells but also to repress mitotic functions in the endoreduplicating cells of the female germ line, which have repeated S and G phases. Characterization of the phenotype of imaginal discs arrested (ida) mutants indicate that the APC5 subunit encoded by this gene is not required for sister chromatid separation, leading the suggestion that other APC/C subunits meet this requirement (Bentley, 2002). The functions of the APC/C components Cdc27 and Cdc16 of Drosophila have been tested individually by RNA interference (RNAi) in cultured Drosophila S2 cells (Huang, 2002), suggesting that the two molecules might modulate the activity of the complex in different ways at different sites. Huang's experiments indicated a tendency for sister centromeres to separate following cdc27 RNAi but not cdc16 RNAi, with chromatids appearing more scattered on the spindle. However, there was also a greater requirement for cdc27 function for chromosome-associated cyclin A degradation. One difficulty in interpreting RNAi experiments with S2 cells is their ability, under some growth conditions, to continue cycling. Therefore, the phenotypes of mutations in mákos (mks), the Drosophila gene encoding the counterpart of Cdc27, were examined in larval neuroblasts. Such mutant cells show a robust mitotic arrest with chromosomes in a metaphase-like arrangement and a failure to degrade mitotic cyclins. The finding of some degree of sister chromatid separation in mks supports the growing evidence for differential roles for APC subunits for different mitotic functions (Deak, 2003 and references therein).

The APC/C is activated upon entry into mitosis and its substrate specificity is thought to be regulated mainly by the transient association of co-factors such as Cdc20/Fizzy and Cdh1/Hct1/Fzr (see Drosophila Fizzy related). APC/C function is further known to be regulated by phosphorylation, for which Cdk1/cyclin-B, Polo-like kinase 1 (Plk1) and cAMP-dependent protein kinases have all been implicated as playing major roles. Plk1 appears to facilitate APC/C activation by preferentially phosphorylating Cdc16 and Cdc23, and indeed its fission yeast counterpart, Plo1, binds specifically to the Cdc23 (APC8) subunit. Although Polo-like kinases have been reported to be required for APC/C activation, mks mutants have been found to enhance the mutant phenotype of an hypomorphic allele of polo with respect to its ability to orchestrate one known function of Polo-like kinases: the recruitment of gamma-tubulin and associated molecules to the centrosome (Deak, 2003 and references therein).

Should the spindle function aberrantly, activity of the APC/C is prevented by the spindle integrity checkpoint until chromosomes are correctly aligned at metaphase. One of the spindle integrity checkpoint proteins, Mad2, also binds the APC/C as part of the mechanism that prevents its activity until chromosomes are correctly aligned on a functional spindle. In mks mutants, the spindle checkpoint protein Bub1 is present at the kinetochores of prometaphase and metaphase chromosomes but the metaphase arrest of mks mutants cannot be over-run by mutation in bub1. The 55 kDa regulatory subunit of protein phosphatase 2A (PP2A) has also been implicated in regulating Cdk1 activity and the APC/C regulated process of sister chromatid separation at the metaphase-anaphase transition in budding yeast and has been postulated to have a spindle checkpoint function. This study shows that mks induced metaphase arrest can be overcome by mutations in the 55 kDa regulatory subunit of PP2A (Drosophila Twins), identifying an alternative means of initiating anaphase. It is possibile that inactivation of the 55 kDa regulatory subunit of PP2A is one target of the APC/C in relation to the regulatory networks governing mitotic progression (Deak, 2003).

The mitotic arrest seen in the mks mutant neuroblasts appears to be more robust than the effects seen by Huang (2002), who interfered with Drosophila cdc27 function in cultured cells by RNAi. Thus, the observations with mks mutants strengthen and extend several findings from this previous study. Although the mks mutant cells accumulate both A- and B-type mitotic cyclins, several lines of evidence indicate that their sister chromatids have undergone separation. These include in situ hybridization studies with a centromeric satellite sequence, and immunostaining experiments to reveal a centromeric antigen and the checkpoint protein Bub1 curiously at separated kinetochores. Several aspects of the mks phenotype thus resemble those seen in mutations in ida, the Drosophila gene encoding the APC5 homolog. Both mutants show an elevated mitotic index with cells arrested in a metaphase-like state yet with separated sister chromatids in the continued presence of Bub1 at kinetochores and elevated levels of B-type cyclin. The observation of sister chromatid separation in ida mutants has led to the suggestion that removal of the single APC5 subunit might only affect degradation of a subset of APC/C substrates. In other words, APC5 would not be necessary for the degradation of securin, the release of separase and the cleavage of cohesins, and so chromatid separation would be unaffected. If, as has been suggested by King (1995), Cdc27 is a core component of the APC/C, the finding of sister separation in mks would suggest lack of a widespread requirement for the APC/C for sister chromatid separation in Drosophila, at least to the extent that it is observed in mks and ida mutants. The possibility cannot be excluded that separation occurs as a result of residual function of either of these gene products because no null alleles of ida or mks have been studied. A similar argument can be applied to the RNAi experiments, in which, although the knock-down is impressive, there is some residual protein following the treatment. Alternatively, the minimal catalytic activity of the APC/C, thought to be mediated by the direct ubiquitination of substrates by Apc11 acting alone or together with Apc2 depending upon the E2 enzyme, could be sufficient for securin degradation. It is noted, however, that there are subtle differences between the phenotypes of mks and ida. The proportion of anaphase figures is higher in ida cells and these also exhibit a level of aneuploidy that is not detected in mks. At present, it remains unclear whether this reflects specific characteristics of the mutant alleles studied or whether the block to chromosome movement at anaphase is stronger following perturbation of Cdc27 function (Deak, 2003 and references therein).

The motivation for studying genetic interactions between mks and polo arose from evidence pointing to a role for Polo kinase in APC/C activation. Depleting Plx1 in extracts of Xenopus, blocks degradation of cyclin B. In budding yeast, the Polo-like kinase encoded by CDC5 has also been implicated in activation of the APC/C. Moreover, mammalian APC/C can be activated by Plk1 phosphorylation in vitro. The phosphorylation patterns of APC/C components have been carefully studied; Plk1 and Cdk1/cyclin-B have additive effects in phosphorylating and activating the APC/C; the former preferentially phosphorylates Cdc16 and Cdc23, and the latter preferentially phosphorylates Cdc27. The fission yeast Polo-like kinase Plo1 interacts physically with the Cut23 component of the APC/C. Nevertheless, it appears that the APC/C is active in the polo1 because cyclin A levels are reduced and cyclin B remains, as would be seen following checkpoint delay at metaphase. Only a slightly earlier lethal phase of mks was seen when in combination with different polo alleles. By contrast, the unexpected observation was made that the weakly hypomorphic phenotype of polo1 in larval neuroblasts is enhanced by mks in respect to its effects upon centrosome structure and function (Deak, 2003, and references therein).

The original polo1 allele encodes an apparently full-length protein that is incompletely phosphorylated and has low catalytic activity. Thus, polo1 is a weak hypomorph and, although embryos derived from homozygous polo1 mothers show defects in the recruitment of the centrosomal antigen CP190, those mothers would appear to have been able to develop to adulthood in part because of the lack of any obvious centrosomal defect in mitotically dividing cells of their larval central nervous systems. This is in contrast to brains of the strong hypomorphic alleles polo9 or polo10, in which neuroblasts arrest in a metaphase-like state, with spindles that have core centrosomal components, such as CNN, at their spindle poles but lack gamma-tubulin and CP190. This phenotype echoes the requirement for Polo kinase in recruiting gamma-tubulin to the centrosome upon mitotic entry following experiments in which antibodies against Plk1 were microinjected into HeLa cells (Deak, 2003 and references therein).

The finding that mks enhances the centrosomal phenotype of polo1 such that it resembles the stronger polo hypomorphs suggests that the APC/C is required either directly or indirectly to fully activate Polo kinase function. It is possible to gain a measure of the activity of Polo kinase by assessing the presence of the MPM2 epitope. Indeed, at least one Drosophila centrosomal protein (Asp) has been shown to acquire an MPM2 epitope following phosphorylation by Polo. The finding of the absence of MPM2 reactivity at the centrosome in mks1 polo1 mutants is therefore consistent with reduced Polo kinase activity. A direct functional interaction between the APC/C and Polo cannot be excluded because Polo kinase has been shown to bind the APC/C, at least in fission yeast, and both the APC/C and Polo kinase are present on the centrosome. However, the idea is favored that an early mitotic function of the APC/C might be required for the full activation of Polo kinase and the recruitment of the gamma tubulin ring complex (gamma-TuRC) to the centrosome. Indeed, cyclin A is normally degraded ahead of cyclin B and continues to be degraded when cells are arrested at the spindle checkpoint by microtubule depolymerizing drugs, but remains present in both mks1 and mks1 polo1 cells. Normally, the degradation of cyclin A takes place from the beginning of mitosis in an APC/C-dependent manner. Thus, it is postulated that because APC/C is normally active from the very beginning of mitosis, one of its functions might be to facilitate the full activation of Polo kinase at the centrosome by mediating the degradation either of cyclin A or some other crucial inhibitory molecule. This would constitute a new cell cycle autoregulatory loop whereby the early mitotic functions of the APC/C are required for the activation of an enzyme itself implicated in activating later functions of the APC/C (Deak, 2003).

The metaphase-like arrest of mks cells cannot be overcome by the bub1 mutation. This is consistent with known functions of the APC/C downstream of the spindle integrity checkpoint. However, the ability of mutants in the aar/twins gene to overcome the metaphase arrest of mks is suggestive of an alternate mechanism for regulating the transition. In fact, the aar/twins mutant appears to be totally epistatic to (functioning downstram of) mks. Thus, the mks aar/twins double mutant shows a similar proportion of anaphase figures to the aar/twins mutant alone, and this is higher than the frequency of anaphases seen in wild-type cells. These observations cast some light on the possible multiple functions of the regulatory subunit of PP2A encoded by aar/twins in regulating anaphase and mitotic exit. It suggests that APC/C function might normally be required to inactivate the wild-type 55 kDa PP2A subunit that, in turn, negatively regulates sister chromatid separation. Thus, in the absence of aar/twins function, this aspect of APC/C involvement would not be required for anaphase, thus accounting for the epistasis of aar/twins to mks. The mutant aar/twins phenotype that then develops is akin to that observed following the expression of non-degradable forms of cyclin B, in which mitosis proceeds into anaphase. This outcome would be reinforced by a failure to exit mitosis as a result of the reduced ability of aar/twins mutants to dephosphorylate substrates of Cdk1 (Deak, 2003).

The anaphases in aar/twins and in the double mutant are highly abnormal, indicating that the checkpoint pathway that monitors chromosome alignment at metaphase and works through regulation of the APC/C is being circumvented. Consequently, there are many bridging and lagging chromatids in both aar/twins and mks aar/twins anaphase figures. This phenotype bears a striking resemblance to that seen in mutants of the CDC55 gene of budding yeast that encodes the orthologous regulatory subunit of PP2A. Cells with a cdc55 mutation have also been shown to leave mitosis without B-type cyclin destruction, in this case apparently owing to inhibitory tyrosine phosphorylation. However, it is also postulated in budding yeast that Cdc55p function is required for the kinetochore/spindle checkpoint. Such cdc55 mutants are sensitive to nocodazole and, in contrast to the situation for Drosophila cells, cdc55 mutations do not overcome the arrest imposed by mutation in an APC/C protein, in this case Cdc23p. Nevertheless, the abnormal morphology of cdc55 mutants and their conditional lethality is suppressed by a cdc28F19 mutation that encodes a variant kinase not susceptible to inhibitory phosphorylation. By contrast, nocodazole sensitivity cannot be suppressed by cdc28F19. This suggests that, in yeast, Cdc55p might have a checkpoint role that is independent of Cdc28/Cdk1 and a second role in regulating Cdc28 phosphorylation (Deak, 2003).

At the present time, it is not possible to account for how the APC/C might regulate the function of the 55kDa PP2A subunit although one possibility is through its direct proteolysis. It appears that this regulatory subunit of PP2A must participate in regulating the metaphase-anaphase transition, in controlling the activity of Cdk1 and in dephosphorylating Cdk1 substrates. It therefore remains a question of considerable future interest to determine exactly how these activities are coordinated (Deak, 2003).

Cyclin B3 activates the Anaphase-Promoting Complex/Cyclosome in meiosis and mitosis

In mitosis and meiosis, chromosome segregation is triggered by the Anaphase-Promoting Complex/Cyclosome (APC/C), a multi-subunit ubiquitin ligase that targets proteins for degradation, leading to the separation of chromatids. APC/C activation requires phosphorylation of its APC3 and APC1 subunits, which allows the APC/C to bind its co-activator Cdc20. The identity of the kinase(s) responsible for APC/C activation in vivo is unclear. Cyclin B3 (CycB3) is an activator of the Cyclin-Dependent Kinase 1 (Cdk1) that is required for meiotic anaphase in flies, worms and vertebrates. It has been hypothesized that CycB3-Cdk1 may be responsible for APC/C activation in meiosis but this remains to be determined. Using Drosophila, this study found that mutations in CycB3 genetically enhance mutations in tws, which encodes the B55 regulatory subunit of Protein Phosphatase 2A (PP2A) known to promote mitotic exit. Females heterozygous for CycB3 and tws loss-of-function alleles lay embryos that arrest in mitotic metaphase in a maternal effect, indicating that CycB3 promotes anaphase in mitosis in addition to meiosis. This metaphase arrest is not due to the Spindle Assembly Checkpoint (SAC) because mutation of mad2 that inactivates the SAC does not rescue the development of embryos from CycB3-/+, tws-/+ females. Moreover, CycB3 was found to promote APC/C activity and anaphase in cells in culture. CycB3 physically associates with the APC/C, is required for phosphorylation of APC3, and promotes APC/C association with its Cdc20 co-activators Fizzy and Cortex. These results strongly suggest that CycB3-Cdk1 directly activates the APC/C to promote anaphase in both meiosis and mitosis (Garrido, 2020).

Mitosis and meiosis (collectively referred to as M-phase) are distinct modes of nuclear division resulting in diploid or haploid products, respectively. In animals, both require the breakdown of the nuclear envelope, the condensation of chromosomes and their correct attachment on a microtubule-based spindle, where chromosomes are under tension and chromatids are held together by cohesins. Progression through these initial phases requires multiple phosphorylation events of various protein substrates by mitotic kinases including Cyclin-Dependent Kinases (CDKs) activated by their mitotic cyclin partners. M-phase completion from this point (mitotic exit) requires the degradation of mitotic cyclins, and the dephosphorylation of several mitotic phosphoproteins by phosphatases including Protein Phosphatase 2A (PP2A). Mitotic exit begins with the segregation of chromosomes in anaphase. In mitosis, sister chromatids segregate. In meiosis I, replicated homologous chromosomes segregate, and in the subsequent meiosis II, sister chromatids segregate. Nuclear divisions are completed with the reassembly of a nuclear envelope concomitant with the decondensation of chromosomes. How mitosis and meiosis are alike and differ in the molecular mechanisms of their exit programs is not completely understood (Garrido, 2020).

Chromosome segregation is triggered by the Anaphase-Promoting Complex/Cyclosome (APC/C), a multi-subunit E3 ubiquitin ligase. By catalysing the addition of ubiquitin chains on the separase inhibitor securin, the APC/C targets it for degradation by the proteasome. As a result, separase cleaves cohesins, allowing separated chromosomes to migrate towards opposing poles of the spindle. Activation of the APC/C in mitosis requires its recruitment of its co-factor Cdc20. This recruitment can be prevented by the Spindle-Assembly Checkpoint (SAC), a complex mechanism that allows the sequestration of Cdc20 until all chromosomes are correctly attached on the spindle. Cdc20 binding to the APC/C is also inhibited by its phosphorylation at CDK sites. Phosphatase activity is then required to dephosphorylate Cdc20 and allow its binding of the APC/C for its activation of anaphase. In addition, phosphorylation of the APC/C itself is required to allow Cdc20 binding. Phosphorylation of APC3/Cdc27 and APC1 is key to this process. Phosphorylation of APC3 at CDK sites promotes the subsequent phosphorylation of APC1, inducing a conformational change in APC1 that opens the Cdc20 binding site. However, the precise identity of the kinase(s) involved in this process in vivo is unknown (Garrido, 2020).

At least 3 types of cyclins contribute to M-phase in animals: Cyclins A, B and B3. The Cyclin A type (A1 and A2 in mammals) can activate Cdk1 or Cdk2 and is required for mitotic entry, at least in part by allowing the phosphorylation of Cdc20 to prevent its binding and activation of the APC/C. This allows mitotic cyclins to accumulate without being ubiquitinated prematurely by the APC/C and degraded. The Cyclin B type (B1 and B2 in mammals) also promotes mitotic entry and is required for mitotic progression by allowing the phosphorylation of several substrates by Cdk1. Mammalian Cyclin B3, which can associate with both Cdk1 and Cdk2, is required for meiosis but its contribution to mitosis is less clear in view of its low expression in somatic cells. Drosophila possesses a single gene for each M-phase cyclin: CycA (Cyclin A), CycB (Cyclin B) and CycB3 (Cyclin B3) that collaborate to ensure mitotic progression by activating Cdk1. Genetic and RNAi results suggest that they act sequentially, CycA being required before prometaphase, CycB before metaphase and CycB3 at the metaphase-anaphase transition. CycA is the only essential cyclin, as it is required for mitotic entry. CycB and CycB3 mutants are viable, but mutations of CycB and CycB3 are synthetic-lethal, suggesting redundant roles in mitosis. However, mutation of CycB renders females sterile due to defects in ovary development, and mutant males are also sterile (Garrido, 2020).

Drosophila CycB3 associates with Cdk1 and is required for female meiosis (Jacobs, 1998). In Drosophila, eggs normally stay arrested in metaphase I of meiosis until egg laying triggers entry into anaphase I and the subsequent meiosis II. However, CycB3 mutant eggs predominantly stay arrested in meiosis I (Bourouh, 2016). In addition, silencing CycB3 expression in early embryos delays anaphase onset during the syncytial mitotic divisions (Yuan, 2015). Cyclin B3 is also required for anaphase in female meiosis of vertebrates and worms. In mice, RNAi Knock-down of Cyclin B3 in oocytes inhibits the metaphase-anaphase transition in meiosis I. Recently, two groups independently knocked out the Cyclin B3-coding Ccnb3 gene in mice and found that they were viable but female-sterile due to a highly penetrant arrest in meiotic metaphase I. In C. elegans, the closest Cyclin B3 homolog, CYB-3 is required for anaphase in meiosis and mitosis (Garrido, 2020).

How Cyclin B3 promotes anaphase in any system is unknown. One possibility is that it is required for Cdk1 to phosphorylate the APC/C on at least one of its activating subunits, APC3 or APC1. This has not been investigated. Another possibility is that inactivation of Cyclin B3 leads to an early mitotic defect that activates the SAC. This appears to be the case in C. elegans, because inactivation of the SAC rescues normal anaphase onset in the absence of CYB-3. However, in Drosophila, inactivation of the SAC by the mutation of mad2 did not eliminate the delay in anaphase onset observed when CycB3 is silenced in syncytial embryos. Similarly, in mouse oocytes, silencing Mad2 does not rescue the meiotic metaphase arrest upon Cyclin B3 depletion. In other studies, SAC markers on kinetochores did not persist in metaphase-arrested Ccnb3 KO oocytes, and SAC inactivation by chemical inhibition of Mps1 did not restore anaphase. Finally, it is also possible that Cyclin B3 is required upstream of another event required for APC/C activation, for example the activation of a phosphatase required for Cdc20 dephosphorylation and subsequent recruitment to the APC/C (Garrido, 2020).

This study has investigated how CycB3 promotes anaphase in Drosophila. Several lines of evidence are reported indicating that CycB3 directly activates the APC/C in both meiosis and mitosis (Garrido, 2020).

Altogether, the results strongly suggest that CycB3-Cdk1 directly activates the APC/C by phosphorylation, promoting its function at the metaphase-anaphase transition in meiosis and in both maternally driven early embryonic mitoses and somatic cell divisions. This regulation is likely mediated by the phosphorylation in the activation loop of APC3 by CycB3-Cdk1 that ultimately promotes the recruitment of the Cdc20-type co-activators Fizzy and Cortex. Previous work has shown that APC3 phosphorylation and APC/C activation by cyclin-CDK complexes require their CKS subunit (see Cks30A). CKS subunits can act as processivity factors that bind phosphorylated sites to promote additional phosphorylation by the CDK. Thus, phosphorylation of APC3 would prime the binding of a cyclin-CDK-CKS complex to promote the additional phosphorylation of APC1, allowing for Cdc20 binding. It has been shown that mutation of phosphorylation sites into Asp or Glu residues cannot substitute for the presence of phosphate in the CKS binding site. Therefore, it was not possible to generate a mutation in APC3 that would have mimicked phosphorylation at S316 to enhance cyclin-CDK-CKS binding. Such a mutation in APC3, if it were possible, would have potentially rescued APC/C activity in the absence of CycB3 according to this model. However, it is likely that this analysis did not detect all phosphorylation sites in the APC/C. Thus, the possibility cannot be exclustion that other phosphorylation events, mediated by CycB3-Cdk1 or another kinase, may be required for complete APC/C activation. For example, other phosphorylation events have been proposed to regulate APC/C localization. It is even formally possible that CycB3-Cdk1 is required to activate another proline-directed kinase that phosphorylates APC3 at S316. The interdependence between CycB3 and Tws that this study uncovered may reflect a role of PP2A-Tws in the recruitment of Cdc20 co-activators to the APC/C. Cdc20 must be dephosphorylated at CDK sites before binding the APC/C, and in human cells both PP2A-B55 and PP2A-B56 promote this event (Garrido, 2020).

CycB3 is strongly required for APC/C activation in meiosis and in the early syncytial mitoses, and to a lesser extent in other mitotic divisions, despite the presence of two additional mitotic cyclins, CycA and CycB, capable of activating Cdk1. There are many possible reasons for this requirement. Overexpression of stabilized forms of CycA or CycB can block or slow down anaphase, suggesting that they may interfere with APC/C function in this transition. However, under normal expression levels, CycA or CycB or both may contribute to activate the APC/C like CycB3. CycB3 mutant flies develop until adulthood, which implies that the APC/C can be activated to induce anaphase in at least a vast proportion of mitotic cells, and this activation could be mediated by CycA and/or CycB. CycA is essential for viability and CycB mutants show strong female germline development defects, complicating the examination of potential roles for these cyclins at the metaphase-anaphase transition. Thus, in principle, the requirements for CycB3 in female meiosis, in embryos and in mitotic cells in culture could merely reflect the need for a minimal threshold of total mitotic cyclins. This possibility is considered unlikely because CycB3 is expressed at much lower levels than CycB in early embryos. Moreover, while maternal heterozygosity for mutations in CycB3 and tws causes a metaphase arrest in embryos, heterozygosity for mutations in CycB and tws does not cause embryonic defects. In fact, genetic results suggest that the function of CycB is antagonized by PP2A-Tws in embryos, while CycB3 and PP2A-Tws collaborate for APC/C activation in embryos. Thus, although it is possible that CycA and CycB can participate in APC/C activation, CycB3 probably has some unique feature that makes it particularly capable of promoting APC/C activation (Garrido, 2020).

By what mechanism could CycB3 be particularly suited for APC/C activation? Cyclins can play specific roles by contributing to CDK substrate recognition or by directing CDK activity in space and time. This study did not investigate the precise nature of the molecular recognition of the APC/C by CycB3. It may be that CycB3 possesses a specific binding site for the APC/C that is lacking in CycA and CycB. Another possibility is that differences in localization between cyclins dictate their requirements. In particular, while CycA and CycB are cytoplasmic in interphase, CycB3 is nuclear. It is surmised that the nuclear localization of CycB3 may help concentrate CycB3 in the spindle area upon germinal vesicle breakdown, when the very large oocyte enters meiosis. In future studies, it will be interesting to compare the ability of different mitotic cyclins to activate the APC/C and to determine the molecular basis of potential differences (Garrido, 2020).

In any case, the results show that CycB3 activates the APC/C and that this regulation is essential in Drosophila. Cyclin B3 has been shown to be required for anaphase in female meiosis of insects (Drosophila), worms (C. elegans) and vertebrates (mice). It is tempting to conclude that the activation of the APC/C is a function of Cyclin B3 conserved in all these species. However, in C. elegans embryos, the metaphase arrest upon CYB-3 (Cyclin B3) inactivation requires SAC activity. The underlying mechanism and whether it also occurs in other systems remain to be determined. However, CYB-3 plays roles in C. elegans that have not been detected for Cyclin B3 in flies or vertebrates, including a major role in mitotic entry, where CYB-3 mediates the inhibitory phosphorylation of Cdc20. In this regard, C. elegans CYB-3 may be more orthologous to Cyclin A. Yet, given that Cyclin B3 is required for anaphase in a SAC-independent manner in flies and mice, it seems reasonable to suggest that the direct activation of the APC/C by Cyclin B3 is conserved in vertebrates (Garrido, 2020).


The P element insertions of mks1and mks2 were mapped to the left arm of chromosome 3, to location 65E7-12. The elements appear to be directly responsible for the observed phenotypes, because mutants could revert to wild type following P element remobilization. Molecular cloning of genomic DNA flanking the P element insertion sites enabled isolation of embryonic cDNA clones. An additional full-length cDNA clone was subsequently obtained from the Berkeley Drosophila Genome Project. Sequence analysis of the mákos locus and its corresponding cDNAs identified an open reading frame of ~3 kb interrupted by two small introns. The P element insertion of mks1 is located within the 5'-untranslated region 253 bp upstream of the ATG initiator codon. The mks2 P-element insertion is 532 bp upstream of the ATG. RT-PCR experiments reveal a dramatic reduction of the mks transcript in both alleles, with mks1 being a strong hypomorph. A genomic DNA fragment containing the transcription unit was cloned into a P-element germ-line transformation vector and was found to be able to rescue mks mutants. Moreover, a variant of this DNA segment in which ~1 kb of internal sequence is deleted fails to rescue the mutant (Deak, 2003).

cDNA clone length - 3033

Bases in 5' UTR - 270

Exons - 3

Bases in 3' UTR - 135


Amino Acids - 900

Structural Domains

The sequence of mks reveals that it encodes the Drosophila homolog of the budding yeast gene CDC27, a subunit of the APC/C. The MKS protein contains ten tandemly arranged 34 amino acid repeat units termed TPR motifs, nine of which are located in a single block at the carboxy-terminal with a single disconnected unit closer to the N-terminal end. This arrangement is conserved in CDC27 homologs from yeasts to humans (Tugendreich, 1995). Such TPR repeats have been implicated in facilitating protein-protein interactions between APC/C subunits (Deak, 2003).

Cdc27: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 25 August 2022

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