Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA
* Author for correspondence (e-mail: gschupbach{at}molbio.princeton.edu)
Accepted 13 June 2005
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Cks, Drosophila, Cell cycle, Meiosis
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 and Dunphy, 1996;
Patra and Dunphy, 1998
;
Patra et al., 1999
;
Spruck et al., 2003
).
Mammalian Cks2 and C. elegans CKS-1 are also required for the
metaphase/anaphase transition (Polinko and
Strome, 2000
; Spruck et al.,
2003
). Cks1 in mammals plays a seemingly unrelated role in
promoting S-phase progression as an adaptor protein that links the
SCFSkp2 complex to one of its substrates, the Cdk inhibitor p27
(Ganoth et al., 2001
;
Spruck et al., 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 et al., 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.
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, we analyzed a collection
of maternal effect mutants (Schupbach and
Wieschaus, 1989) that arrest early in embryogenesis. We 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 we present evidence that this is through an
effect on the activity of Cortex, a germline-specific adaptor for the
Anaphase-Promoting Complex (APC). We 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.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunoprecipitations and western blots
Immunoprecipitations were performed on frozen 0-8 hour embryos or 0-4 hour
embryos from females carrying a single copy of nanos-Gal4VP16
(Van Doren et al., 1998) and a
single copy of a given FLAG-tagged Cks in 10 volumes of 50 mmol/l Tris (pH 8),
150 mmol/l NaCl, 1 mmol/l CaCl2 and 0.1% Tween 20 supplemented with
complete mini protease inhibitor (Roche) and type I and II phosphatase
inhibitors (Sigma). Anti-FLAG beads and FLAG peptide for elution were from
Sigma. Antiserum against Drosophila Cks30A was raised in guinea pig
(Pocono Rabbit Farms) against full-length His-tagged Cks30A expressed in
bacteria, and was affinity purified against GST-tagged Cks30A. This was used
at 1/100 on western blots. Other antibodies for western blots were rabbit
-Cdk2 at 1/500, rabbit
-Fzr (1/500) and rabbit
-Cyclin B3
(1/1000) (all gifts from Christian Lehner), mouse
-Fzy (from Ian
Dawson), mouse
-Cyclin B and
-Cyclin A (both from Developmental
Studies Hybridoma Bank) at 1/10, mouse
-FLAG M5 (Sigma) at 1/20 and
rabbit
-PSTAIR (Santa Cruz) at 1/1000. Relative Cyclin A levels were
determined by loading 1x, 4x, 8x, 12x and 16x
dilutions of Cks30AKO extracts and comparing band
intensity (using IP Image software) to 1x of wild-type extract.
Antibody staining and fluorescence in-situ hybridization
To visualize early events in female meiosis, mature oocytes from
hand-dissected ovaries were activated in vitro
(Page and Orr-Weaver, 1997).
Later stages of meiosis were observed in fixed embryos from 0-20 minute
collections. For most embryo stainings, timed collections were dechorionated
in bleach, fixed in 1:1 heptane/methanol. For phospho-histone H3 staining,
embryos were fixed in 3.7% formaldehyde in PBST/heptane. Embryos and ovaries
were mounted in tetrahydronapthelene after incubating in methanol followed by
isopropanol. Antibodies were rat
-Tubulin (Cappel) at 1/200, rabbit
-Cnn (gift of Thomas Kaufman) at 1/500, mouse
-Lamin Dmo
(Development Studies Hybridoma Bank) at 1/20, rabbit
-phospho-histone
H3 (Upstate) at 1/500, rabbit
-Caspase 3 (Cell Signaling Technologies)
at 1/500. DNA was visualized with Oligreen (Molecular Probes) at 1/500.
Fluorescence in-situ hybridization (FISH) against X- and Y-chromosome-specific
repeat sequences was performed according to Dernburg
(Dernburg, 2000
).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
In wild type, after completion of meiosis, the three post-meiotic nuclei that do not join with the male pronucleus undergo nuclear envelope breakdown and arrest in a metaphase-like state. Chromosomes are condensed and label for the prophase/metaphase marker Phospho-Histone H3, and are associated with a radial aster of microtubules (Fig. 2I). In rem, chromosomes condensed and stained positive for phospho-histone H3, but the polar body microtubule aster did not form, and instead a shell of microtubules surrounded the chromatin (Fig. 2J).
|
|
Cks30AKO embryos also displayed a pronounced delay in entry into mitosis. In a 0-20 minute collection of embryos, only 29% had mitotic spindles, compared with 74% in wild type (Table 1). Instead, they typically contained two separated microtubule asters, one of which was associated with condensed chromatin (Fig. 4B, arrow; Table 1). In many cases, a thin spindle connected the two asters (data not shown), suggesting that these were centrosomes that had duplicated in advance of mitosis but had not yet assembled a mitotic spindle. In 20-40 minute collections, 54% of embryos had assembled a mitotic spindle, and in 40-80 minute collections, 80% of embryos had a single mitotic spindle. The others appeared to arrest with separated centrosomes that underwent a small number of extra divisions (Fig. 4C). Therefore, Cks30AKO is delayed in, or arrests before, mitotic spindle assembly. This delay is apparent but less pronounced in CksS30AP61 embryos (Table 1).
Cks30AKO causes a delay in exit from metaphase II of meiosis
As in Cks30AP61, meiosis in
Cks30AKO progressed normally up until the resumption of
meiosis following egg activation, and a similar phenotype was observed in
which the tandem meiosis II spindle was kinked or separated. In addition,
Cks30AKO mutants displayed a pronounced delay in metaphase
of meiosis II (Fig. 4B, Table 1).
Cks30AP61 mutants also displayed a slight delay in
completing meiosis (Table 1).
The delayed exit from female meiosis in Cks30AKO embryos
would be predicted to result in a failure to complete nuclear fusion. Indeed,
FISH to X and Y chromosomes revealed that most embryos (11/12) did not
complete joining of the male and female pronuclei
(Fig. 4D). The occasional
failure to complete nuclear fusion in Cks30AP61 embryos
could similarly be explained by the observed partial delay in exit from female
meiosis (Table 1).
Cks30A interacts genetically with Cdk1 in histoblast cells
In addition to a maternal effect lethal phenotype, all cks30A
mutants have a defect in abdominal cuticle deposition indicative of a partial
disruption of larval abdominal histoblast development
(Fig. 5A,B). This phenotype was
variable, but was expressed in all alleles, and at similar strengths. In wild
type, the abdominal histoblasts arrest in G2 of the cell cycle through most of
larval development and then re-enter the cell cycle after puparium formation.
In Cdk1 and escargot mutants, histoblasts fail to arrest in
G2 and instead enter an endocycle
(Hayashi, 1996;
Hayashi et al., 1993
).
Myb mutants produce a similar phenotype, although these specifically
disrupt resumption of mitosis in pupae
(Katzen et al., 1998
). To
determine if either process is disrupted in Cks30A, we looked for
genetic interactions between Cks30A and Cdk1 and between
Cks30A and myb. While reducing myb activity in a
Cks30A mutant background did not produce a stronger phenotype (data
not shown), Cks30A in combination with loss of a single copy of
Cdk1 produced a very strong effect
(Fig. 5C). Similarly, a weak
allele of Cdk1 displayed a mild abdominal phenotype at 18°C, but
this was strongly enhanced by loss of a single copy of Cks30A (data
not shown). These genetic interactions suggest that Cks30A interacts with Cdk1
to prevent polyploidy in larval histoblasts.
|
|
|
The two mutant alleles, Cks30AHG24 and
Cks30ARA74 disrupt an amino acid, P61, that lies in a
region of the protein implicated in Cdk binding
(Bourne et al., 1996). To
determine if the loss of function phenotype in these alleles could be due to a
failure to bind to Cdks, we constructed a FLAG-Cks30AP61L mutant
(corresponding to the mutation in Cks30ARA74). As
expected, this failed to rescue the Cks30AKO mutant
(Table 2). We then performed
immunoprecipitations with FLAG-Cks30AP61L and found that it was
strongly impaired for Cdk binding, supporting the idea that
Drosophila Cks30A functions with Cdks to promote female meiotic
progression and mitotic exit in embryos.
Cks proteins all share an anion-binding domain that can interact with the
phospho-epitope Ser/pSer/pThr/X. This domain can interact with partially
phosphorylated Cdk substrates and has been suggested to recruit them to the
Cdk to allow more phosphorylations to occur. To test the significance of this
domain, we altered two residues in the domain
(Bourne et al., 2000). The
mutant FLAG-Cks30AK10ES50E completely failed to rescue a
Cks30AKO mutant (Table
2), indicating that the anion-binding domain is essential for
Cks30A function. As expected, this mutation had no effect on Cdk interaction
(data not shown).
Cks30A is required for Cyclin A destruction
Cdk1 activity during mitosis is necessary for the activation of the APC. In
the cellularized Drosophila embryo, the APC targets the mitotic
Cyclins A, B and B3 sequentially for degradation, and this degradation is
necessary for mitotic exit (Sigrist et
al., 1995). APC-mediated Cyclin degradation has also been
implicated in anaphase progression in the early syncytial divisions
(Su et al., 1998
;
Huang and Raff, 1999
).
Therefore, the metaphase arrest in Cks30A mutants could be due to a
requirement for Cks30A in the Cdk1-dependent activation of the APC.
To test this possibility, we examined cyclin levels in
Cks30AKO mutants. Cyclins B and B3 occurred at wild-type
levels in Cks30A mutants, but the levels of Cyclin A were much higher
than in wild type (Fig. 7A).
This was not due to a secondary effect of cell cycle arrest, as it was also
observed in ovaries from Cks30A mutants
(Fig. 7A). It was also not due
to increased transcription, as cyclin A mRNA levels were equivalent
in wild-type and Cks30A mutants
(Fig. 7B). In cellularized
embryos, the introduction of non-degradable Cyclin A results in a metaphase
delay or arrest (Sigrist et al.,
1995
), and therefore it is likely that the metaphase arrest in
Cks30A mutants was a result of the failure to degrade Cyclin A. If
so, it may be possible to relieve the requirement for Cks30A by
reducing Cyclin A levels. Indeed, when Cyclin A gene dosage was
halved, we saw a significant rescue of Cks30A mutants
(Fig. 7C).
The destruction of mitotic cyclins is mediated by the APC and an associated
subunit, CDC20, that is thought to target the APC to its substrates (reviewed
by Peters, 2002). In
Drosophila, three CDC20 homologs, Fzy, Fzr (Rap) and Fzr2 have known
roles in cyclin degradation in vivo (Dawson
et al., 1995
; Jacobs et al.,
2002
; Sigrist et al.,
1995
; Sigrist and Lehner,
1997
). Of these, only Fzy appears be active in the syncytial
embryo (Jacobs et al., 2002
;
Raff et al., 2002
). Like
Cks30A mutants, hypomorphic alleles of fzy result in a
metaphase arrest in the first mitotic division of the embryo
(Dawson et al., 1993
),
suggesting the possibility that Cks30A regulates Cyclin A levels through the
APCFzy. However, we found that Cyclin A levels were not affected in
ovaries or embryos from fzy mutant females (data not shown). Based on
homology, the cortex gene encodes a fourth, distantly related
Drosophila CDC20 protein (Chu et
al., 2001
). Mutations in cortex result in a meiotic
phenotype similar to that in Cks30A, in which meiosis is arrested at
metaphase of the second division
(Lieberfarb et al., 1996
;
Page and Orr-Weaver, 1996
). We
examined cyclin levels in cortex mutant ovaries and found that, as in
Cks30A, those of Cyclin A but not Cyclins B and B3 were elevated
(Fig. 7D and data not shown).
Therefore, Cks30A may regulate maternal Cyclin A levels through an
effect on the activity of APCCortex.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cks30A interacts with Cdk1 to promote spindle assembly and anaphase progression
Cks30A mutants displayed a strikingly simple mitotic phenotype:
most embryos from mutant females arrested in metaphase of the first mitotic
division. Similarly, Cks30A mutants displayed 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.
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, our FISH experiments indicate that this mitotic
arrest occurs even in embryos that successfully underwent pronuclear fusion.
Also, mutations in -Tubulin67C that block pronuclear fusion do
not lead to a mitotic arrest in the embryo
(Komma and Endow, 1995
),
indicating that these events are not coupled.
In addition to a crucial role in exit from mitosis, Cks30A is important in at least one aspect of entry into mitosis: spindle formation. Cks30AKO mutants were 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.
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 (Patra and Dunphy,
1998). Xenopus Cks2 is also required in vitro for entry
into mitosis (Patra and Dunphy,
1996
), 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 (Spruck et al.,
2003
), while in C. elegans cks-1 is not required for
entry into anaphase, but is necessary for proper chromosome segregation in
meiosis I (Polinko and Strome,
2000
), 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.
The two roles for Cks30A appear to reflect a conserved role in
promoting Cdk1 activity (reviewed by
Pines, 1996). 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 (reviewed by
Nigg, 2001
). We have found
that in Drosophila Cks30A and Cdk1 interact in vivo, and that
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, our genetic and physical
evidence supports the conclusion that the observed interaction with Cdk1 is
required for Cks30A function.
Cks30A-Cdk1 may activate the Cortex-APC-mediated destruction of Cyclin A
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. First, a similar metaphase arrest is seen in cellularized
embryos expressing non-degradable Cyclin A
(Sigrist et al., 1995), while
syncytial embryos with a slight excess of Cyclin A (approximately 1-3x
wild type) due to mutations in grapes display a metaphase delay
(Su et al., 1999
). Second, 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 (Page and Orr-Weaver,
1996
; Lieberfarb et al.,
1996
), and we have found that cortex is also required for
Cyclin A destruction in the ovary. These results argue that Cks30A and the
APCCortex function in the same pathway leading to Cyclin A
destruction, although we cannot rule out the possibility that Cks30A and
Cortex act in independent pathways to promote Cyclin A destruction.
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 (Patra and
Dunphy, 1996; Patra and
Dunphy, 1998
; Patra et al.,
1999
). 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
(Kraft et al., 2003
).
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.
Cyclin destruction in nuclear divisions versus cell divisions
In mammalian cells and in the cellularized embryo, the completion of
mitosis depends on the sequential destruction of the three mitotic cyclins by
the APCFzy (Sigrist et al.,
1995). Cyclin A is destroyed first in prometaphase, dependent on
APCFzy activity. Although the APCFzy is active, the
spindle checkpoint is thought to inhibit its activity on Cyclin B. Upon
spindle assembly, the checkpoint is relieved and APCFzy can mediate
Cyclin B destruction (Peters,
2002
).
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)
(Edgar et al., 1994), 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 (Huang and Raff,
1999
; Su et al.,
1998
). 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 (Su et al.,
1998
). Our results suggest that this metaphase arrest is due to
the failure of APCCortex-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
(Edgar et al., 1994
). Despite
this difference, it remains possible that local oscillations in Cyclin A and
Cyclin B could drive these syncytial cell cycles.
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 APCFzy is responsible for the
sequential destruction of all three mitotic cyclins
(Dawson et al., 1995;
Sigrist et al., 1995
). 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 APCCortex and if the APCCortex 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 (Lieberfarb et al.,
1996
; Page and Orr-Weaver,
1996
), 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 (Polinko and Strome,
2000
; Spruck et al.,
2003
), 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), as cortex mutants,
either alone or in combination with Cks30A, have no effect on
abdominal development (Page and
Orr-Weaver, 1996
; Lieberfarb
et al., 1996
) (A.S. and T.S., unpublished).
Cks proteins in specialized cell cycles
A specific requirement for Cks30A in activation of the maternal-specific
APCCortex would explain why Cks30A is essential for
anaphase progression in female meiosis and the syncytial embryo but not in
most cell types. We can rule out an alternative possibility, that Cks30A is
functionally redundant with the other Drosophila Cks, Cks85A.
Cks85A mutants alone or in combination with Cks30A, do not
have obvious defects in exit from mitosis (A.S. and T.S., unpublished).
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, we
conclude 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
(Giot et al., 2003). Two
residues on Cks1 have recently been found to be crucial for Skp2 binding in
vitro (Seeliger et al., 2003
),
and these residues are conserved or similar in Drosophila Cks85A (see
Fig. 3A). If indeed Cks85A
represents the Drosophila Cks1 ortholog, it is perhaps not surprising
that Cks30A cannot functionally replace Cks85A, as it has been found that Cks2
orthologs cannot bind Skp2 in vitro
(Ganoth et al., 2001
;
Spruck et al., 2001
). 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
(Spruck et al., 2003
). The
failure to rescue Cks30A mutant phenotypes cannot be due to an
inability of Cks85A to interact with Cdks, as 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.
In conclusion, we find that 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.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bartek, J. and Lukas, J. (2001). p27 destruction: Cks1 pulls the trigger. Nat. Cell Biol. 3, E95-E98.[CrossRef][Medline]
Bourne, Y., Watson, M. H., Hickey, M. J., Holmes, W., Rocque, W., Reed, S. I. and Tainer, J. A. (1996). Crystal structure and mutational analysis of the human CDK2 kinase complex with cell cycle-regulatory protein CksHs1. Cell 84,863 -874.[CrossRef][Medline]
Bourne, Y., Watson, M. H., Arvai, A. S., Bernstein, S. L., Reed, S. I. and Tainer, J. A. (2000). Crystal structure and mutational analysis of the Saccharomyces cerevisiae cell cycle regulatory protein Cks1: implications for domain swapping, anion binding and protein interactions. Structure Fold Des. 8, 841-850.[CrossRef][Medline]
Chu, T., Henrion, G., Haegeli, V. and Strickland, S. (2001). Cortex, a Drosophila gene required to complete oocyte meiosis, is a member of the Cdc20/fizzy protein family. Genesis 29,141 -152.[CrossRef][Medline]
Dawson, I. A., Roth, S., Akam, M. and Artavanis-Tsakonas, S.
(1993). Mutations of the fizzy locus cause metaphase arrest in
Drosophila melanogaster embryos. Development
117,359
-376.
Dawson, I. A., Roth, S. and Artavanis-Tsakonas, S. (1995). The Drosophila cell cycle gene fizzy is required for normal degradation of cyclins A and B during mitosis and has homology to the CDC20 gene of Saccharomyces cerevisiae. J. Cell Biol. 129,725 -737.[Abstract]
Dernburg, A. (2000). In Situ Hybridization to Somatic Chromosomes. In Drosophila Protocols (ed. W. Sullivan M. Ashburner and R. S. Hawley), pp. 25-55. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
Edgar, B. A., Sprenger, F., Duronio, R. J., Leopold, P. and O'Farrell, P. H. (1994). Distinct molecular mechanism regulate cell cycle timing at successive stages of Drosophila embryogenesis. Genes Dev. 8,440 -452.[Abstract]
Foe, V. E., Odell, G. M. and Edgar, B. A. (1993). Mitosis and morphogenesis in the Drosophila embryo. In The Development of Drosophila Melanogaster (ed. M. Bate and A. Martinez-Arias), pp. 149-300. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
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.[CrossRef][Medline]
Giot, L., Bader, J. S., Brouwer, C., Chaudhuri, A., Kuang, B.,
Li, Y., Hao, Y. L., Ooi, C. E., Godwin, B., Vitols, E. et al.
(2003). A protein interaction map of Drosophila melanogaster.
Science 302,1727
-1736.
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]
Hayashi, S. (1996). A Cdc2 dependent checkpoint
maintains diploidy in Drosophila. Development
122,1051
-1058.
Hayashi, S., Hirose, S., Metcalfe, T. and Shirras, A. D.
(1993). Control of imaginal cell development by the escargot gene
of Drosophila. Development
118,105
-115.
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.[CrossRef][Medline]
Huang, J. and Raff, J. W. (1999). The
disappearance of cyclin B at the end of mitosis is regulated spatially in
Drosophila cells. EMBO J.
18,2184
-2195.
Jacobs, H., Richter, D., Venkatesh, T. and Lehner, C. (2002). Completion of mitosis requires neither fzr/rap nor fzr2, a male germline-specific Drosophila Cdh1 homolog. Curr. Biol. 12,1435 -1441.[CrossRef][Medline]
Katzen, A. L., Jackson, J., Harmon, B. P., Fung, S. M., Ramsay,
G. and Bishop, J. M. (1998). Drosophila myb is
required for the G2/M transition and maintenance of diploidy. Genes
Dev. 12,831
-843.
Komma, D. J. and Endow, S. A. (1995). Haploidy
and androgenesis in Drosophila. Proc. Natl. Acad. Sci.
USA 92,11884
-11888.
Kraft, C., Herzog, F., Gieffers, C., Mechtler, K., Hagting, A.,
Pines, J. and Peters, J. M. (2003). Mitotic regulation
of the human anaphase-promoting complex by phosphorylation. EMBO
J. 22,6598
-6609.
Lane, M. E. and Kalderon, D. (1993). Genetic investigation of cAMP-dependent protein kinase function in Drosophila development. Genes Dev. 7,1229 -1243.[Abstract]
Lee, L. A. and Orr-Weaver, T. L. (2003). Regulation of cell cycles in Drosophila development: intrinsic and extrinsic cues. Annu. Rev. Genet. 37,545 -578.[CrossRef][Medline]
Lieberfarb, M. E., Chu, T., Wreden, C., Theurkauf, W., Gergen,
J. P. and Strickland, S. (1996). Mutations that
perturb poly(A)-dependent maternal mRNA activation block the initiation of
development. Development
122,579
-588.
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.[CrossRef]
Nigg, E. A. (2001). Mitotic kinases as regulators of cell division and its checkpoints. Nat. Rev. Mol. Cell. Biol. 2,21 -32.[CrossRef][Medline]
Page, A. W. and Orr-Weaver, T. L. (1996). The
Drosophila genes grauzone and cortex are necessary for proper female meiosis.
J. Cell Sci. 109,1707
-1715.
Page, A. W. and Orr-Weaver, T. L. (1997). Activation of the meiotic divisions in Drosophila oocytes. Dev. Biol. 183,195 -207.[CrossRef][Medline]
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.[Abstract]
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.
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.
Peters, J. M. (2002). The anaphase-promoting complex: proteolysis in mitosis and beyond. Mol. Cell 9, 931-943.[CrossRef][Medline]
Pines, J. (1996). Cell cycle: reaching for a role for the Cks proteins. Curr. Biol. 6,1399 -1402.[CrossRef][Medline]
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.[CrossRef][Medline]
Raff, J. W., Jeffers, K. and Huang, J. Y.
(2002). The roles of Fzy/Cdc20 and Fzr/Cdh1 in regulating the
destruction of cyclin B in space and time. J. Cell
Biol. 157,1139
-1149.
Rong, Y. S., Titen, S. W., Xie, H. B., Golic, M. M., Bastiani,
M., Bandyopadhyay, P., Olivera, B. M., Brodsky, M., Rubin, G. M. and
Golic, K. G. (2002). Targeted mutagenesis by homologous
recombination in D. melanogaster. Genes Dev.
16,1568
-1581.
Schupbach, T. and Wieschaus, E. (1989). Female
sterile mutations on the second chromosome of Drosophila melanogaster. I.
Maternal effect mutations. Genetics
121,101
-117.
Seeliger, M. A., Breward, S. E., Friedler, A., Schon, O. and Itzhaki, L. S. (2003). Cooperative organization in a macromolecular complex. Nat. Struct. Biol. 10,718 -724.[CrossRef][Medline]
Sigrist, S. J. and Lehner, C. F. (1997). Drosophila fizzy-related down-regulates mitotic cyclins and is required for cell proliferation arrest and entry into endocycles. Cell 90,671 -681.[CrossRef][Medline]
Sigrist, S., Jacobs, H., Stratmann, R. and Lehner, C. F. (1995). Exit from mitosis is regulated by Drosophila fizzy and the sequential destruction of cyclins A, B and B3. EMBO J. 14,4827 -4838.[Abstract]
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.[CrossRef][Medline]
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.
Stern, B., Ried, G., Clegg, N. J., Grigliatti, T. A. and Lehner,
C. F. (1993). Genetic analysis of the Drosophila cdc2
homolog. Development
117,219
-232.
Su, T. T., Sprenger, F., DiGregorio, P. J., Campbell, S. D. and
O'Farrell, P. H. (1998). Exit from mitosis in
Drosophila syncytial embryos requires proteolysis and cyclin degradation, and
is associated with localized dephosphorylation. Genes
Dev. 12,1495
-1503.
Su, T. T., Campbell, S. D. and O'Farrell, P. H. (1999). Drosophila grapes/CHK1 mutants are defective in cyclin proteolysis and coordination of mitotic events. Curr. Biol. 9,919 -922.[CrossRef][Medline]
Van Doren, M., Broihier, H. T., Moore, L. A. and Lehmann, R. (1998). HMG-CoA reductase guides migrating primordial germ cells. Nature 396,466 -469.[CrossRef][Medline]
Related articles in Development:
|