InteractiveFly: GeneBrief

Cyclin-dependent kinase subunit 30A: Biological Overview | References

Cks is a small highly conserved protein that plays an important role in cell cycle control in different eukaryotes. Cks proteins have been implicated in entry into and exit from mitosis, by promoting Cyclin-dependent kinase (Cdk) activity on mitotic substrates. In yeast, Cks can promote exit from mitosis by transcriptional regulation of cell cycle regulators. Cks proteins have also been found to promote S-phase via an interaction with the SCF^Skp2 Ubiquitination complex. The Drosophila Cks gene, Cks30A (corresponding to the gene remnants), is required for progression through female meiosis and the mitotic divisions of the early embryo through an interaction with Cdk1 (Cdc2). Cks30A mutants are compromised for Cyclin A destruction, resulting in an arrest or delay at the metaphase/anaphase transition, both in female meiosis and in the early syncytial embryo. Cks30A appears to regulate Cyclin A levels through the activity of a female germline-specific anaphase-promoting complex, CDC20-Cortex. A second closely related Cks gene, Cks85A, plays a distinct, non-overlapping role in Drosophila, and the two genes cannot functionally replace each other (Swan, 2005).

Passage through the cell cycle must be precisely regulated during development. A number of conserved cell cycle regulators have been shown to act at important developmental transitions in various organisms, including Drosophila. Cks, or Suc1, is a small cell cycle regulator that was first identified by its ability to interact genetically and physically with the Cyclin-dependent kinases (Cdks) in yeasts (Hadwiger, 1989; Hayles, 1986). Members of this family were subsequently found in many multicellular organisms, including Xenopus, Caenorhabditis elegans and humans (reviewed by Bartek, 2001; Pines, 1996). In addition to interacting with Cdks, Cks proteins share an anion-binding domain implicated in binding to the phospho-epitope Ser/pSer/pThr/X, found on many mitotic proteins; and a domain that mediates a conformational switch between a monomeric and a dimeric form in vitro (Swan, 2005 and references therein).

Despite the strong conservation of these proteins, studies in different animal models have revealed a surprising number of distinct roles at different points in the cell cycle. Studies of the Cks2 homolog in Xenopus (Xe-p9) indicate a role in the G2-M transition and the metaphase/anaphase transition, possibly by linking the Cdk to its substrates (Patra, 1996; Patra, 1998; Patra, 1999; Spruck, 2003). Mammalian Cks2 and C. elegans CKS-1 are also required for the metaphase/anaphase transition (Polinko, 2000; Spruck, 2003). Cks1 in mammals plays a seemingly unrelated role in promoting S-phase progression as an adaptor protein that links the SCF^Skp2 complex to one of its substrates, the Cdk inhibitor p27 (Ganoth, 2001; Spruck, 2001). Recent work in yeast has revealed yet another function for Cks. Saccharomyces cerevisiae Cks1 was found to recruit the 19S and 20S proteosome to the promoter of the CDC20 gene to promote its transcription late in mitosis (Morris, 2003). In metazoans, which all appear to have two Cks genes, it is not yet clear how the two Cks genes carry out these diverse roles, and to what degree there is functional redundancy between them (Swan, 2005 and references therein).

Female meiosis in Drosophila represents an excellent system for studying the developmental control of cell cycle progression. Female meiosis progresses through well-characterized transitions that are linked to development of the oocyte. With the aim of identifying genes required for completion of female meiosis in Drosophila, this study analyzed a collection of maternal effect mutants that arrest early in embryogenesis. It was found that the remnants (rem) gene is required for female meiosis and for mitosis in the early embryo, and that this gene corresponds to the Drosophila Cks gene at cytological location 30A (Cks30A). In addition to its maternal role, Cks30A appears to function with Cdk1 to prevent S-phase in G2-arrested histoblasts in the larva. Cks30A interacts with Cdk1/cyclin complexes, and this interaction is necessary for its function. One of the key functions of Cks30A is to mediate the destruction of Cyclin A, and evidence is presented that this is through an effect on the activity of Cortex, a germline-specific adaptor for the Anaphase-Promoting Complex (APC). It was also found that a closely related Cks gene, Cks85A, plays a distinct role in Drosophila, and the two genes cannot substitute for each other in vivo (Swan, 2005).

The maternal effect lethal gene, remnants, corresponds to one of the two Drosophila Cks genes (Cks30A). Analysis of two hypomorphic alleles and a null allele made by homologous recombination confirmed that Cks30A is not essential for cell cycle regulation in most tissue types. Rather, Cks30A functions in specialized cell cycles: the abdominal histoblast divisions, female meiosis and the syncytial divisions of the early embryo (Swan, 2005).

Cks30A mutants displayed a strikingly simple mitotic phenotype: most embryos from mutant females arrested in metaphase of the first mitotic division. Similarly, Cks30A mutants display a pronounced delay or arrest in metaphase of female meiosis II. Therefore, there is a common requirement for Cks30A in metaphase to anaphase progression in female meiosis II and in early embryonic mitosis. The second meiotic division is similar to a mitotic division in that it involves the segregation of sister chromatids, and therefore Cks30A may be part of a conserved machinery that is required for both of these processes (Swan, 2005).

An alternative explanation for the mitotic arrest in Cks30A mutants is that it is a secondary effect of a prior failure in meiosis or pronuclear fusion. However, FISH experiments indicate that this mitotic arrest occurs even in embryos that successfully undergo pronuclear fusion. Also, mutations in alpha-Tubulin67C, that block pronuclear fusion, do not lead to a mitotic arrest in the embryo, indicating that these events are not coupled (Swan, 2005).

In addition to a crucial role in exit from mitosis, Cks30A is important in at least one aspect of entry into mitosis: spindle formation. Cks30A^KO mutants are severely delayed in assembly of the first mitotic spindle. Cks30A was also required for proper assembly of the female meiotic spindle and the specialized spindle-like microtubule aster of the polar bodies. Therefore, Cks30A appears to be required at two points in the mitotic (or meiotic) cell cycle: in prometaphase for spindle assembly and at the metaphase-to-anaphase transition (Swan, 2005).

These dual roles for Cks30A in meiosis appear to be at least partially conserved in other metazoans. Xenopus Cks2 (Xe-p9), like Cks30A, is required for the metaphase-to-anaphase transition in meiosis II. Xenopus Cks2 is also required in vitro for entry into mitosis, and this may be related to the in vivo requirement for Cks30A in spindle assembly in meiosis and mitosis. Cks genes in other eukaryotes also appear to have related but distinct functions in meiosis. Mouse cks2 is essential for anaphase progression in meiosis I of both male and female meiosis, while in C. elegans cks-1 is not required for entry into anaphase, but is necessary for proper chromosome segregation in meiosis I, possibly reflecting a role in meiotic spindle assembly. Therefore, Cks genes appear to share a common requirement in entry into and exit from meiosis in different eukaryotes (Swan, 2005).

The two roles for Cks30A appear to reflect a conserved role in promoting Cdk1 activity. Cdk1 is the central mitotic Cdk, and its kinase activity on specific mitotic proteins is required for entry into mitosis, including spindle assembly, and in maintaining the metaphase state. Cdk1 activity is also required for exit from mitosis through activation of the APC, which in turn promotes anaphase progression by targeting mitotic cyclins for destruction. In Drosophila Cks30A and Cdk1 interact in vivo, and mutations in Cks30A that disrupt Cdk1 binding are compromised for activity in vivo. Cks30A also interacts genetically with Cdk1 in another cell type, the abdominal histoblasts. Therefore, genetic and physical evidence supports the conclusion that the observed interaction with Cdk1 is required for Cks30A function (Swan, 2005).

Cks30A is required for the destruction of Cyclin A in the ovary and in the syncytial embryo and two observations argue that it is this failure to degrade Cyclin A that results in the observed delay or arrest in metaphase of meiosis II and in mitosis: (1) a similar metaphase arrest is seen in cellularized embryos expressing non-degradable Cyclin A, while syncytial embryos with a slight excess of Cyclin A (approximately 1-3 x wild type) due to mutations in grapes display a metaphase delay; (2) the Cks30A mutant phenotype can be partially rescued by lowering Cyclin A levels. The CDC20 homolog, cortex, is also required for exit from meiosis II, and cortex is also required for Cyclin A destruction in the ovary. These results argue that Cks30A and the APC^Cortex function in the same pathway leading to Cyclin A destruction, although the possibility that Cks30A and Cortex act in independent pathways to promote Cyclin A destruction cannot be ruled out (Swan, 2005).

In vitro studies of Cks2 in Xenopus have led to a model in which Cks bound to Cdk1 recruits phosphorylated Cdk1 substrates to the kinase, allowing these substrates to be more efficiently recognized and thereby further phosphorylated by Cdk1. The CDC27 and CDC16 components of the APC are key targets of Cks-Cdk1 phosphorylation in Xenopus. While it is not yet clear how APC phosphorylation leads to its activation, there is evidence that one of the effects of phosphorylation is to stimulate CDC20 binding to the APC. Therefore it is possible that in Drosophila Cks30A-Cdk1 phosphorylates the APC, and this phosphorylation specifically stimulates the association of Cortex with the APC. Alternatively, Cks30A-Cdk1 may directly phosphorylate and activate Cortex (Swan, 2005).

In mammalian cells and in the cellularized embryo, the completion of mitosis depends on the sequential destruction of the three mitotic cyclins by the APC^Fzy. Cyclin A is destroyed first in prometaphase, dependent on APC^Fzy activity. Although the APC^Fzy is active, the spindle checkpoint is thought to inhibit its activity on Cyclin B. Upon spindle assembly, the checkpoint is relieved and APC^Fzy can mediate Cyclin B destruction (Swan, 2005).

It now appears that some but not all aspects of anaphase progression are conserved in the second meiotic division and in the nuclear divisions of the syncytial embryo. Although overall Cyclin B levels do not oscillate during the early syncytial divisions (cycles 1 to 8), Cyclin B appears to undergo local degradation on the mitotic spindle at anaphase, and the injection of stabilized Cyclin B into syncytial embryos results in an early anaphase arrest. By contrast to the early anaphase arrest upon Cyclin B stabilization, the injection of an APC-inhibiting peptide into early embryos results in a metaphase arrest. The results suggest that this metaphase arrest is due to the failure of APC^Cortex-mediated Cyclin A destruction. Like Cyclin B levels, Cyclin A levels do not oscillate detectably in cycles 1 to 7, although unlike Cyclin B, this appears to be due to a balance between constant destruction and new protein synthesis. Despite this difference, it remains possible that local oscillations in Cyclin A and Cyclin B could drive these syncytial cell cycles (Swan, 2005).

While the importance of cyclin destruction may be conserved in the early embryo, the means by which the cyclins are destroyed appears to be different. In cellularized embryos, the APC^Fzy is responsible for the sequential destruction of all three mitotic cyclins. In the syncytial embryo, Fzy is not required for Cyclin A destruction. Cortex, a diverged, female germline-specific CDC20, targets Cyclin A for destruction, but has no detectable effect on Cyclin B or B3 levels in the syncytial embryo. It remains possible that Cortex is responsible for the destruction of local pools of maternal Cyclin B (and possibly B3). Alternatively, the known maternal requirement for fzy may reflect a role in the local destruction of these cyclins. This would suggest a model in which the germline utilizes two CDC20 homologs, Cortex and Fzy, to mediate the sequential destruction of Cyclins A, B and possibly B3 in the syncytial embryo. Further work will be needed to test this model. It is also not clear if Cyclin A is the only target of the APC^Cortex and if the APC^Cortex is the only target of Cks30A-Cdk1. In addition to metaphase arrest, Cks30A mutants have spindle assembly delays or defects, a phenotype that has not been observed in other cell types to result from a failure to degrade Cyclin A. Interestingly, cortex mutants also have abnormal meiosis II spindles and fail to assemble a mitotic spindle around the male pronucleus, suggesting the possibility that the sole function of Cks30A in the female germline is to activate Cortex. Like Cks30A, C. elegans cks-1 and mouse cks2 appear to be predominantly required for meiosis, and this may also reflect specific roles in activating meiosis-specific APC complexes. The histoblast requirement for Cks30A, in contrast, is unlikely to represent a role in Cortex activation (or subsequent Cyclin A destruction), since cortex mutants, either alone or in combination with Cks30A, have no effect on abdominal development (Swan, 2005).

A specific requirement for Cks30A in activation of the maternal-specific APC^Cortex would explain why Cks30A is essential for anaphase progression in female meiosis and the syncytial embryo but not in most cell types. An alternative possibility, that Cks30A is functionally redundant with the other Drosophila Cks, Cks85A, cannot be ruled out. Cks85A mutants alone or in combination with Cks30A, do not have obvious defects in exit from mitosis. Furthermore, while closely related to Cks30A, Cks85A cannot replace Cks30A when expressed in the female germline, and Cks30A cannot replace Cks85A when expressed zygotically. Therefore, it is concluded that the two Drosophila Cks genes have distinct and non-overlapping functions. Recently, Cks85A was found to interact with a Drosophila Skp2 homolog in a genome-wide yeast two-hybrid screen. Two residues on Cks1 have recently been found to be crucial for Skp2 binding in vitro, and these residues are conserved or similar in Drosophila Cks85A. If indeed Cks85A represents the Drosophila Cks1 ortholog, it is perhaps not surprising that Cks30A cannot functionally replace Cks85A, since it has been found that Cks2 orthologs cannot bind Skp2 in vitro. However, it is unexpected that Cks85A cannot substitute for Cks30A in vivo. To date all Cks proteins tested can stimulate the Cdk-dependent phosphorylation of mitotic proteins in vitro, and the mouse Cks1 can functionally replace Cks2 in vivo. The failure to rescue Cks30A mutant phenotypes cannot be due to an inability of Cks85A to interact with Cdks, since Cks85A binds Cdks with even greater affinity than does Cks30A. It is possible that, analogous to the Cks1/Skp2 interaction, Cks30A has an as-yet-to-be-identified partner that is necessary for its mitotic activities. Cks85A would, therefore, be unable to carry out the mitotic activities because of an inability to bind this putative Cks30A partner (Swan, 2005).

In conclusion, Drosophila Cks30A is crucial for Cdk1 activity in spindle assembly and anaphase progression in female meiosis and early embryonic mitosis, and at least part of this activity appears to be to regulate Cyclin A levels. Cks30A functions non-redundantly with another closely related Drosophila cks, Cks85A (Swan, 2005).

The Cdc20 (Fzy)/Cdh1-related protein, Cort, cooperates with Fzy and Cks30A in cyclin destruction and anaphase progression in meiosis I and II in Drosophila

Meiosis is a highly specialized cell division that requires significant reorganization of the canonical cell-cycle machinery and the use of meiosis-specific cell-cycle regulators. The anaphase-promoting complex (APC, a machine for degrading proteins; see APC subunits Cdc27 and morula; for review see Acquaviva, 2006) and a conserved APC adaptor/activator, Cdc20 (also known as Fizzy), are required for anaphase progression in mitotic cells. The APC has also been implicated in meiosis, although it is not yet understood how it mediates these non-canonical divisions. Cortex (Cort) is a diverged Fzy homologue that is expressed in the female germline of Drosophila, where it functions with the Cdk1-interacting protein Cks30A to drive anaphase in meiosis II. This study shows that Cort functions together with the canonical mitotic APC adaptor Fzy to target the three mitotic cyclins (A, B and B3) for destruction in the egg and drive anaphase progression in both meiotic divisions. In addition to controlling cyclin destruction globally in the egg, Cort and Fzy appear to both be required for the local destruction of cyclin B on spindles. Cyclin B associates with spindle microtubules throughout meiosis I and meiosis II, and dissociates from the meiotic spindle in anaphase II. Fzy and Cort are required for this loss of cyclin B from the meiotic spindle. These results lead to a model in which the germline-specific APC^Cort cooperates with the more general APC^Fzy, both locally on the meiotic spindle and globally in the egg cytoplasm, to target cyclins for destruction and drive progression through the two meiotic divisions (Swan, 2007).

Cks30A belongs to a highly conserved family of proteins that bind to and stimulate the activity of the mitotic kinase Cdk1. In Xenopus, the Cks30A homologue Xep9 stimulates the Cdk-dependent phosphorylation of APC subunits, and thereby promotes the activation of the APC^Fzy complex (Patra, 1998). The current results suggest that Cks30A may have a similar role in stimulating both the APC^Fzy and APC^Cort in female meiosis in Drosophila. (1) Cks30A, like cort and fzy, is required for the completion of meiosis II and, like fzy, it is required for the completion of the first mitotic division of embryogenesis. (2) Cks30A, as are Cort and Fzy, is necessary for global cyclin destruction in the Drosophila egg and for local cyclin B destruction on the meiotic spindle. Global levels of cyclin A and cyclin B3 are elevated to a greater extent in Cks30A mutants than in single mutants for cort or fzy, consistent with the idea of Cks30A activating both Cort and Fzy. (3) Cks30A is necessary for the activity of ectopically expressed Cort in the adult wing. Cks30A may also play a role in activating APC^Fzy in mitotic cells. the temperature-sensitive fzy⁶ allele is lethal at all temperatures in a Cks30A-null background, suggesting that the Cks30A-dependent activation of APC^Fzy becomes essential when Fzy activity is compromised (Swan, 2007).

Although Cks30A appears to promote the activity of the APC^Cort and the APC^Fzy, these complexes seems to retain some activity in the absence of Cks30A. Whereas cort and fzy cause an arrest in meiosis II, Cks30A-null mutants are typically delayed only in meiosis II (Swan, 2005). Also, although cyclin A and cyclin B3 levels are elevated more in Cks30A eggs than in either fzy or cort, their levels are still not as high as in fzy; cort double mutants, indicating that Fzy and Cort can destroy cyclin A and cyclin B3 to some degree in the absence of Cks30A. Cyclin B destruction is even less dependent on Cks30A, because cyclin B levels are affected less in Cks30A mutants than in either cort or fzy single mutants. Therefore, Cks30A may be more crucial for the activity of APC^Cort and APC^Fzy complexes on cyclin A and cyclin B3, and less crucial for their activity on cyclin B. The relatively weaker meiotic arrest in Cks30A mutants compared to fzy; cort double mutants may also indicate that the APC has other meiotic targets that can be destroyed in the absence of Cks30A (Swan, 2007).

A pre-anaphase role for a Cks/Suc1 in acentrosomal spindle formation of Drosophila female meiosis

Conventional centrosomes are absent from a female meiotic spindle in many animals. Instead, chromosomes drive spindle assembly, but the molecular mechanism of this acentrosomal spindle formation is not well understood. This study screened female sterile mutations for defects in acentrosomal spindle formation in Drosophila female meiosis. One of them, remnants (rem), disrupted bipolar spindle morphology and chromosome alignment in non-activated oocytes. It was found that rem encodes a conserved subunit of Cdc2 (Cks30A). Since Drosophila oocytes arrest in metaphase I, the defect represents a new Cks function before metaphase-anaphase transition. In addition, it was found that the essential pole components, Msps and D-TACC, are often mislocalized to the equator, which may explain part of the spindle defect. The second cks gene cks85A, in contrast, has an important role in mitosis. In conclusion, this study describes a new pre-anaphase role for a Cks in acentrosomal meiotic spindle formation (Pearson, 2005).

Spindle formation in female meiosis is unique in terms of the absence of conventional centrosomes. Instead, chromosomes have a central role in the assembly of spindle microtubules. This acentrosomal (also called acentriolar or anastral) spindle formation is common in female meiosis for many animals including mammals, insects and worms. Despite potential medical implications, this spindle formation is much less studied than centrosome-mediated spindle formation in mitosis (Pearson, 2005).

Drosophila provides a valuable tool to study the acentrosomal spindle formation in vivo. Unlike many other species, mature non-activated Drosophila oocytes arrest in metaphase of meiosis I until ovulation, which coincides with fertilization. This provides a unique opportunity to study spindle formation, without interference from chromosome segregation or meiotic exit (Pearson, 2005).

Two components of acentrosomal spindle poles, Msps and D-TACC, physically interact and are crucial for spindle bipolarity (Cullen, 2001). Other studies have identified essential components for spindle formation, such as kinesin-like proteins (Ncd and Sub, γ-tubulin, and a membrane protein surrounding the spindle (Axs). Some of these spindle components are probably modulated by cell-cycle regulators, but knowledge of the regulation is limited. To identify essential components and regulators, a cytological screen was performed for mutants defective in acentrosomal spindle formation of non-activated oocytes (Pearson, 2005).

Through the screen, remnants was identified and identified as a mutant of a Drosophila Cks/Suc1 homologue, Cks30A. Cks is the third subunit of the Cdc2 (Cdk1)-cyclin B complex, but the role of Cks is less clearcut than that of other subunits of the complex. It is implicated in entry into mitosis/meiosis, metaphase-anaphase transition, exit from mitosis/meiosis and inactivation of Cdk inhibitors (Patra, 1996; Polinko, 2000; Ganoth, 2001; Spruck, 2001, Spruck, 2003). This study shows that Cks30A is required for spindle morphogenesis and chromosome alignment in the metaphase I spindle in arrested mature oocytes. This requirement of a Cks before metaphase-anaphase transition represents a new function that has not previously been identified. Furthermore, it was found that essential spindle pole components Msps and D-TACC mislocalize in the mutant, which may be partly responsible for the spindle defects (Pearson, 2005).

For molecular analysis of the acentrosomal spindle in Drosophila female meiosis, female sterile mutants were screened for spindle defects in non-activated oocytes. Female sterile mutants on the second chromosome have previously been isolated. This study focused on classes of mutants that lay eggs that do not develop beyond the blastoderm stage. This category of mutants includes known meiotic mutants affecting spindle formation, such as fs(2)TW1 (γ-tubulin 37C) and subito (a kinesin-like protein (Pearson, 2005).

The identity of the remnants (rem) gene was identified by positional cloneing. The rem gene was previously mapped to 30A-C using a deficiency Df(2L)30AC (Schupbach, 1989). One missense mutation was identified in the gene CG3738 (cks, hereafter called cks30A; Finley, 1994). There were no other mutations within coding sequences and splicing junctions in the region. In addition, the amount and size of the transcripts that are known to be expressed in adult females was tested, and no differences were found between rem and wild type (Pearson, 2005).

Cks30A is one of two Drosophila homologues of Saccharomyces cerevisiae Cks1/Schizosaccharomyces pombe Suc1, a conserved subunit of the Cdc2 (Cdk1)/cyclin B complex, and has been shown to interact with Cdc2 (Finley, 1994). The mutation in rem¹ results in a conversion of the 61st amino acid from proline to leucine. This proline is completely conserved among all Cks homologues, further confirming that the mutation is not a polymorphism. Crystal structure analysis has indicated that this residue forms part of the interaction surface with Cdc2 (Bourne, 1996). Immunoblots using an anti-human Cks1 antibody indicated that this mutation disrupts the stability of the Cks30A protein (Pearson, 2005).

To explain the role of Cks30A, focus was placed on the rem¹ mutant in non-activated oocytes, which arrest in metaphase I. Non-activated oocytes were dissected from wild type and the rem¹ mutant, and chromosomes and spindles were visualized by immunostaining (Pearson, 2005).

In wild type, non-activated mature oocytes contain a single bipolar spindle around chromosomes. Bivalent chromosomes align symmetrically with chiasmatic chromosomes at the equator and achiasmatic chromosomes that are located nearer the poles. The rem¹ mutant was able to enter meiosis, condense chromosomes and assemble microtubules around chromosomes. However, only a minority of spindles showed normal spindle morphology and chromosome alignment (Pearson, 2005).

The most prominent defect in the rem¹ mutant was chromosome misalignment. This defect was observed in about a half of the spindles. Even in the cases in which the spindle remained well organized, chiasmatic chromosomes often moved away from the equator and lost overall symmetrical distribution. The second class of defect in the rem¹ mutant was abnormal spindle morphology. Although the abnormality varied from spindle to spindle in the rem¹ mutant, the most typical defect was the formation of ectopic poles near the spindle equator. The focusing of spindle poles seemed to be unaffected (Pearson, 2005).

Further quantitative analysis showed no significant difference between the phenotypes of rem¹ homozygotes (rem¹/rem¹) and hemizygotes (rem¹/Df). This indicates that the rem¹ mutation is genetically amorphic. A recent independent study (Swan, 2005) has indicated that another weaker allele rem^HG24 (Schupbach, 1989) shows similar abnormalities at a lower frequency. These results indicate that Cks30A is required before the metaphase-anaphase transition for spindle morphology and chromosome alignment (Pearson, 2005).

To gain an insight into the spindle defects in female meiosis, the localization of Msps was examined. Msps protein belongs to a conserved family of microtubule regulators, including XMAP215, and is the first protein identified at the acentrosomal poles in Drosophila (Cullen, 2001; Ohkura, 2001). An msps mutation often leads to the formation of a tripolar spindle in female meiosis I (Pearson, 2005).

In wild type, Msps protein is accumulated at the acentrosomal poles of the metaphase I spindle in female meiosis, although the localization sometimes spreads to the spindle microtubules. In the rem¹ mutant, although the Msps protein is still concentrated at the poles, it is often accumulated around the equator of the spindle. Mislocalization of this important pole protein to the equator in the rem¹ mutant may sometimes lead to the formation of ectopic spindle poles near the equator (Pearson, 2005).

Msps localization is dependent on another pole protein D-TACC, which binds to Msps (Cullen, 2001). To test whether D-TACC also mislocalizes, the localization of D-TACC was examined in the rem¹ mutant. In wild type, D-TACC is highly concentrated at the acentrosomal pole. In the rem¹ mutant, D-TACC often accumulates at the spindle equator, although it is still concentrated around the poles to some degree. In summary, Cks30A is required for correct localization of the essential pole proteins, Msps and D-TACC (Pearson, 2005).

To gain an insight into how the defect in the Cdc2 complex leads to Msps or D-TACC mislocalization to the spindle equator, the localization of cyclin B was examined. Cyclin B is considered to be the main determinant of the activity and cellular localization of the Cdc2 complex. Immunostaining in non-activated oocytes showed that cyclin B is localized to the metaphase I spindle, with a concentration around the spindle equator. This cyclin B localization could suggest a possible regulatory role of the Cdc2 complex in the transport of Msps and D-TACC from the spindle equator to the poles. The cyclin B localization is not affected in the rem mutant, suggesting that Cks30A mainly affects the substrate specificity of the Cdc2 complex, as shown in other systems (Patra, 1998; Pearson, 2005).

The Drosophila genome contains one more predicted cks homologue (CG9790), which is called cks85A. Although mammalian genomes also have two Cks genes, they are more similar in sequence to each other than to either of the two cks genes in Drosophila (Pearson, 2005).

The gene expression pattern of the two cks genes was examined during Drosophila development. RNAs were isolated from various stages of development and analysed by reverse transcription-PCR (RT-PCR) using primers that correspond to each of the cks genes. cks30A gave strong signals in adult females and embryos, whereas it gave only weak signals in adult males, larvae and pupae. This maternal expression pattern is consistent with the observed female sterile phenotype of the cks30A (rem¹) mutant. In contrast, cks85A signals were obtained more uniformly throughout the development without sex specificity in adults. In S2 cultured cells, which originated from embryos, both genes were well expressed (Pearson, 2005).

To identify the Cks proteins, an anti-human Cks1 antibody was used for immunoblots of protein extracts from embryos and S2 cells. Although the antibody recognized many proteins, two bands were detected within a range of molecular weights consistent with the Cks proteins. In embryos laid by the rem¹ mutant, the amount of the smaller band was greatly reduced. To further confirm their identity, S2 cells were subjected to RNA interference (RNAi) using doublestranded RNAs (dsRNAs) corresponding to the cks genes. It was found that both of the bands disappeared when both genes were simultaneously knocked down by RNAi. It indicated that, consistent with RT-PCR results, S2 cells produced both the Cks proteins and that RNAi effectively depletes them (Pearson, 2005).

Cytological analysis showed that cks85A RNAi results in a significant increase in chromosome misalignment/missegregation and spindle abnormality in mitosis after an extended time, whereas cks30A RNAi has a lesser impact on mitotic progression. About a half of anaphase or telophase cells had lagging chromosomes or chromosome bridges after cks85A RNAi. In some cases, spindles contained scattered chromosomes the sister chromatids of which were either attached or detached. The frequency of multipolar spindles was also increased. The genetic and RNAi results indicated that cks85A has an important function in mitotic progression, whereas cks30A mainly functions in female meiosis (Pearson, 2005).

This study has shown a new pre-anaphase function of a Cks protein in acentrosomal spindle formation during Drosophila female meiosis. Through a cytological screen, spindle defects in remnants among female sterile mutants. Cytological analysis showed that Cks30A is required for correct formation of the acentrosomal spindle and chromosome alignment in female meiosis I. The observation on mislocalization of the essential pole components, Msps and D-TACC, in the mutant provides a molecular insight into a role of Cks30A in spindle morphogenesis (Pearson, 2005).

Cks/Suc1 protein is the third subunit of the Cdc2-cyclin B complex, which is conserved across eukaryotes. Although it has been known to be essential for the cell cycle, the function seems to be less straightforward than that of the other subunits of the Cdc2 complex. One reason is that Cks also interacts with other Cdks and has Cdk-independent functions (Ganoth, 2001; Spruck, 2001). Even if Cks is limited to roles in mitosis/meiosis, Cks proteins are implicated in entry into mitosis/meiosis, metaphase-anaphase transition and also exit from mitosis/meiosis (Patra, 1996; Polinko, 2000; Spruck, 2003). Furthermore, the roles of Cks were further complicated by the fact that animal genomes encode two Cks homologues (Pearson, 2005).

Studies in Caenorhabditis elegans and mice showed that one of two cks genes is required for female fertility (Polinko, 2000; Spruck, 2003). Similarly, the results indicated that one of two Drosophila cks homologues, cks30A, is expressed maternally and is required for female meiosis. Further analysis indicated that Cks30A is required for proper bipolar spindle formation and chromosome alignment in mature oocytes arrested in metaphase I. In C. elegans, depletion of one of the Cks proteins by RNAi results in a failure to complete meiosis I (Polinko, 2000). Similarly, in mice, oocytes from a Cks2 knockout cannot progress past metaphase I and a small percentage of oocytes show chromosome congression failure (Spruck, 2003). In both cases, the defects were interpreted mainly as post-metaphase defects. Since Drosophila non-activated oocytes are arrested in metaphase I until ovulation, pre-anaphase function of Cks30A can be distinguised from possible post-metaphase function. This study clearly showed that Drosophila Cks30A has a function in establishing metaphase I, in addition to later functions that have reported recently (Swan, 2005; Pearson, 2005).

At the moment, it is not known how the cks30A mutation disrupts spindle formation and chromosome alignment in female meiosis. It has been thought that a loss of Cks function affects the Cdc2 activity towards certain substrates. It was found that the essential pole components, Msps and D-TACC, mislocalize to the spindle equator in the mutant. Previously, it was hypothesized that Msps is transported by the Ncd motor and anchored to the poles by D-TACC (Cullen, 2001). D-TACC localizes to the poles independently from Ncd, but may also be transported from the spindle equator along microtubules by other motors. Cks30A-dependent Cdc2 activity may be required for activating the transport system at the onset of spindle formation in female meiosis. Consistently, it was found that cyclin B is concentrated around the equator of the metaphase I spindle. Msps is the XMAP215 homologue and belongs to a family of conserved microtubule-associated proteins (Cullen, 1999; Ohkura, 2001). It is a major microtubule regulator, both in mitosis/meiosis and interphase. The mislocalization of this microtubule-regulating activity could lead to the disruption of spindle organization in the mutant (Pearson, 2005).

Search PubMed for articles about Drosophila Cyclin-dependent kinase subunit 30A

Bartek, J. and Lukas, J. (2001). p27 destruction: Cks1 pulls the trigger. Nat. Cell Biol. 3: E95-E98. Medline abstract: 11283628

Bourne, Y., et al. (1996). Crystal structure and mutational analysis of the human CDK2 kinase complex with cell cycle-regulatory protein CksHs1. Cell 84: 863-874. Medline abstract: 8601310

Cullen, C. F. and Ohkura, H. (2001). Msps protein is localized to acentrosomal poles to ensure bipolarity of Drosophila meiotic spindles. Nat Cell Biol 3: 637-642. Medline abstract: 11433295

Finley, R. L. and Brent, R. (1994). Interaction mating reveals binary and ternary connections between Drosophila cell cycle regulators. Proc. Natl. Acad. Sci. 91: 12980-12984. Medline abstract: 7809159

Ganoth, D., Bornstein, G., Ko, T. K., Larsen, B., Tyers, M., Pagano, M. and Hershko, A. (2001). The cell-cycle regulatory protein Cks1 is required for SCF(Skp2)-mediated ubiquitinylation of p27. Nat. Cell Biol. 3: 321-324. Medline abstract: 11231585

Hadwiger, J. A., Wittenberg, C., Mendenhall, M. D. and Reed, S. I. (1989). The Saccharomyces cerevisiae CKS1 gene, a homolog of the Schizosaccharomyces pombe suc1+ gene, encodes a subunit of the Cdc28 protein kinase complex. Mol. Cell. Biol. 9: 2034-2041. Medline abstract: 2664468

Hayles, J., Beach, D., Durkacz, B. and Nurse, P. (1986). The fission yeast cell cycle control gene cdc2: isolation of a sequence suc1 that suppresses cdc2 mutant function. Mol. Gen. Genet. 202: 291-293. Medline abstract: 3010051

Morris, M. C., Kaiser, P., Rudyak, S., Baskerville, C., Watson, M. H. and Reed, S. I. (2003). Cks1-dependent proteasome recruitment and activation of CDC20 transcription in budding yeast. Nature 424: 1009-1013. Medline abstract: 12827207

Ohkura, H., Garcia, M. A. and Toda, T. (2001). Dis1/TOG universal microtubule adaptors-one MAP for all? J. Cell Sci. 114: 3805-3812. Medline abstract: 11719547

Patra, D. and Dunphy, W. G. (1996). Xe-p9, a Xenopus Suc1/Cks homolog, has multiple essential roles in cell cycle control. Genes Dev. 10: 1503-1515. Medline abstract: 8666234

Patra, D. and Dunphy, W. G. (1998). Xe-p9, a Xenopus Suc1/Cks protein, is essential for the Cdc2-dependent phosphorylation of the anaphase-promoting complex at mitosis. Genes Dev. 12: 2549-2559. Medline abstract: 9716407

Patra, D., Wang, S. X., Kumagai, A. and Dunphy, W. G. (1999). The xenopus Suc1/Cks protein promotes the phosphorylation of G(2)/M regulators. J. Biol. Chem. 274: 36839-36842. Medline abstract: 10601234

Pearson, N. J., Cullen, C. F., Dzhindzhev, N. S. and Ohkura, H. (2005). A pre-anaphase role for a Cks/Suc1 in acentrosomal spindle formation of Drosophila female meiosis. EMBO Rep. 6(11): 1058-63. Medline abstract: 16170306

Pines, J. (1996). Cell cycle: reaching for a role for the Cks proteins. Curr. Biol. 6: 1399-1402. Medline abstract: 8939596

Polinko, E. S. and Strome, S. (2000). Depletion of a Cks homolog in C. elegans embryos uncovers a post-metaphase role in both meiosis and mitosis. Curr. Biol. 10: 1471-1474. Medline abstract: 11102813

Schupbach, T. and Wieschaus, E. (1989). Female sterile mutations on the second chromosome of Drosophila melanogaster. I. Maternal effect mutations. Genetics 121: 101-117. Medline abstract: 2492966

Spruck, C., Strohmaier, H., Watson, M., Smith, A. P., Ryan, A., Krek, T. W. and Reed, S. I. (2001). A CDK-independent function of mammalian Cks1: targeting of SCF(Skp2) to the CDK inhibitor p27Kip1. Mol. Cell 7: 639-650. Medline abstract: 11463388

Spruck, C. H., de Miguel, M. P., Smith, A. P., Ryan, A., Stein, P., Schultz, R. M., Lincoln, A. J., Donovan, P. J. and Reed, S. I. (2003). Requirement of Cks2 for the first metaphase/anaphase transition of mammalian meiosis. Science 300: 647-650. Medline abstract: 12714746

Swan, A., Barcelo, G. and Schupbach, T. (2005). Drosophila Cks30A interacts with Cdk1 to target Cyclin A for destruction in the female germline. Development 132: 3669-3678. Medline abstract: 16033797

Swan, A. and Schupbach, T. (2007). The Cdc20 (Fzy)/Cdh1-related protein, Cort, cooperates with Fzy in cyclin destruction and anaphase progression in meiosis I and II in Drosophila. Development 134(5): 891-9. Medline abstract: 17251266

date revised: 17 January 2008 Biological Overview

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