Department of Molecular and Cell Biology, 401 Barker Hall, University of California, Berkeley, CA 94720, USA
* Author for correspondence (e-mail: beckendo{at}uclink.berkeley.edu)
Accepted 8 August 2002
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SUMMARY |
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Key words: Oogenesis, Embryogenesis, Ubiquitin, CSN, Jab1, DNA repair
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INTRODUCTION |
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Recent results have shown that establishment of both AP and DV axes also
depends on the successful repair of DNA double strand breaks (DSBs) that are
formed during meiotic recombination
(Ghabrial et al., 1998;
Ghabrial and Schupbach, 1999
).
Meiotic prophase begins in early region 2a of the germarium, and both
recombination and repair are probably completed before oocyte determination
occurs in region 2b (Huynh and St
Johnston, 2000
).
Meiosis and axis establishment are related to each other because the
accumulation of Grk protein in the oocyte cytoplasm depends on the successful
completion of meiotic recombination
(Ghabrial et al., 1998;
Ghabrial and Schupbach, 1999
).
Mutations in the spindle-class genes, spindle-B
(spn-B), spindle-C (spn-C) and okra
(okr), cause a delay in oocyte determination and a failure to
accumulate Grk protein, leading to defects in AP and DV patterning in late
oogenesis (Gonzalez-Reyes et al.,
1997
; Ghabrial and Schupbach,
1999
). spn-B and okr encode Drosophila
homologs of the RAD51 and RAD54 genes from yeast that are
required for DSB repair (Ghabrial et al.,
1998
; Kooistra et al.,
1997
). Their effects on Grk appear to be mediated by a DNA damage
checkpoint governed by Mei-41, a Drosophila member of the ATM/ATR
family of kinases that are required for DNA damage and recombination
checkpoints in yeast, worms and humans, as well as flies (reviewed by Melo and
Toczysky, 2002; Weinert, 1998
;
Murakami and Nurse, 2000
).
Because they eliminate the checkpoint, mei-41 mutations suppress the
effects of spn or okr mutations. The spn and
okr mutations can also be suppressed by mutations in
mei-W68, which encodes the Drosophila homolog of yeast gene
SPO11, a gene required for the induction of DSBs during recombination
(Ghabrial et al., 1998
;
Roeder, 1997
) These results
indicate that the spn or okr patterning defects result from
activation of a meiotic checkpoint in response to the presence of unrepaired
DSBs.
We show that, like the spindle-class genes, CSN5 is
required for the repair of recombination-induced DSBs during
Drosophila oogenesis. The CSN5 protein (also known as Jab1), is a
subunit of the eight protein COP9 signalosome complex (CSN) originally
identified in plants and conserved from plant to mammalian cells (for reviews,
see Seeger et al., 2001;
Schwechheimer and Deng, 2001
;
Bech-Otschir et al., 2002
). As
the genes for the CSN subunits were identified, a striking similarity was
noticed between them and the eight subunits of the regulatory lid of the
proteasome, suggesting a common ancestry and related function
(Glickman et al., 1998
;
Seeger, 1998; Wei et al.,
1998
). This similarity was intriguing because examinations of CSN
function have shown that it regulates protein stability in pathways leading to
ubiquitination and degradation by the proteasome (reviewed by
Seeger et al., 2001
;
Schwechheimer and Deng, 2001
;
Kim et al., 2001
).
The CSN has been implicated in many regulatory and signaling functions
including activation of the Jun transcription factor, stabilization of nuclear
hormone receptors and interactions with integrins. Most relevant here, the CSN
or its subunits have been shown to regulate multiple steps in the mitotic cell
cycle. For example, the CSN regulates the ubiquitination and degradation of
the CDK inhibitor, p27kip1, and either a small, CSN5-containing
subcomplex or CSN5 alone promotes p27kip1 nuclear export
(Yang et al., 2002;
Tomoda et al., 1999
). In
addition, a CSN-associated kinase activity promotes degradation of p53,
thereby allowing cell cycle progression
(Bech-Otschir et al.,
2001
).
In Drosophila CSN5 is essential for development
(Freilich et al., 1999) and
was recently shown to be required in photoreceptor cells to induce glial cell
migration (Suh et al., 2002
).
We report the first example of a CSN5 effect on meiosis and on axis
determination. We find that homozygous CSN5-mutant clones disrupt
both the DV and AP axes of the oocyte as a result of decreased Grk protein.
These effects on axis determination appear to be caused by activation of the
meiotic recombination checkpoint.
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MATERIALS AND METHODS |
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Genetics
CSN5 homozygous-mutant germline clones were produced by using the
dominant-female-sterile, FLP/FRT technique
(Chou and Perrimon, 1992;
Chou and Perrimon, 1996
).
Females of genotype w; CSN5L4032 P{neoFRT}82B/TM3B, Sb or
w; CSN535ex P{neoFRT}82B/TM3B, Sb were mated with males of
genotype w hsFLP; ovoD1 FRT82B/TM3B, Sb. Their
progeny were heat shocked as third instar larvae or early pupae for two hours
at 37°C for 2 consecutive days to induce FLP expression. Follicle
cell mosaic clones were induced as described by Duffy et al.
(Duffy et al., 1998
): flies
carrying w; P{en2.4-GAL4}e22c P{UAS-FLP1.D}JD1/CyO; P{neoFRT}82B
P{Ubi-GFP(S65T)nls}3R were mated with w; P{neoFRT}82B
CSN5*/TM3B, Sb flies. Eggs were collected and examined for
several days after eclosion. Females were dissected to confirm the presence of
homozygous-mutant follicle cells marked by the absence of GFP.
The original CSN5 P element insertion l(3)L4032 was mobilized by
introducing the P[ry+(2-3)]99B transposase
source (Engels et al., 1987
).
Derivatives that had lost the w+ marker carried by the
original insert were crossed back to CSN5L4032 to identify
imprecise excisions. The majority of new excision lines appear to be precise
excisions of the original P-element insertion. They were fully viable and had
no ovarian defects when homozygous or when heterozygous with
CSN5L4032. Several weak alleles of CSN5 were also
identified. They had poor viability and weak ovarian defects when heterozygous
with CSN5L4032. Finally, several lines failed to
complement the lethality of CSN5L4032, and produced, as
germline clones, similar ovarian defects as did
CSN5L4032.
Staining procedures
The fixation and visualization of egg chambers and embryos was performed as
described (Cant et al., 1994;
Verheyen and Cooley, 1994
).
For immunostaining, the following antibodies were used: mouse anti-Grk (1:20),
rat anti-Grk (1:500), rabbit anti-sperm-tail (1:500), rabbit anti-Vasa
(1:1000) (gifts from T. Schupbach, R. Cohen, T. Karr and P. Lasko). To monitor
lacZ expression of the P-lacZ insertion mutations, ovaries
were treated according to Verheyen and Cooley
(Verheyen and Cooley, 1994
).
For actin visualization, ovaries were stained with rhodamine-conjugated
phalloidin (Molecular Probes). To visualize nuclei, tissues were stained with
DAPI. High magnification fluorescent images were collected on a Zeiss 510
confocal microscope.
In situ hybridization
In situ hybridization using digoxigenin-labeled antisense RNA probes was
carried out as described (Tautz and
Pfeifle, 1989) with modifications
(Harland, 1991
). Hybridization
signals were visualized by histochemical staining with alkaline phosphatase.
Embryos and ovaries were mounted in 70% glycerol and viewed and photographed
with Nomarski optics on a Leica DMRB microscope.
Western and northern blots
Protein extracts for western blot analysis were prepared as described by
Sambrook et al. (Sambrook et al.,
1989). Drosophila CSN5/JAB1 protein was detected using a
mouse polyclonal, and three independent mouse monoclonal, anti-mouse Jab1
antibodies (GeneTex), or a rabbit polyclonal, anti-mouse Jab1 antibody (Santa
Cruz Biotechnology). On a western blot, all of these antibodies recognized the
same 37-38 kDa band, consistent with the predicted size of Drosophila
CSN5. No other specific bands were detected. This band is strongly reduced in
extracts from CSN5L4032 germline clone ovaries and is
reduced to different extents by the hypomorphic alleles derived from
CSN5L4032. Monoclonal antibody MS-JAB11-PXS (GeneTex) was
used for the western blots in this paper. We used rat polyclonal and mouse
monoclonal anti-Grk antibodies (gifts from T. Schupbach and R. Cohen), rabbit
anti-Vasa (a gift from P. Lasko), or monoclonal anti-Actin (ICN). Secondary
antibodies for signal detection were a goat anti-rat or anti-mouse and a
protein-A horseradish peroxidase conjugate (Molecular Probes; Santa Cruz
Biotechnology). Proteins were visualized using chemiluminescent detection (NEN
Life Science Products).
Total or polyA+ RNA was isolated from ovaries as described
(Sambrook et al., 1989). RNA
was resolved on formaldehyde-agarose gels, transferred to nylon membranes,
crosslinked and hybridized by standard procedures.
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RESULTS |
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In embryos, uniformly distributed maternal RNA is evident until gastrulation begins. The earliest zygotic expression is in an anterior stripe during cellular blastoderm. During gastrulation, zygotic expression becomes evident in the ventral furrow, the cephalic furrow, and both the anterior and posterior midgut invaginations (Fig. 1E-H).
CSN5 is required for eggshell patterning
To enable an analysis of early embryonic requirements for CSN5, we
induced homozygous, CSN5-mutant germline clones
(Chou and Perrimon, 1992).
These clones revealed requirements for CSN5 during oogenesis as well
as embryogenesis. In ovarian germline clones the level of CSN5 RNA is
dramatically reduced, but still detectable, indicating that the
P-element-induced allele, CSN5L4032, is hypomorphic
(Fig. 1C,D). Depending on the
paternal allele, embryos derived from the germline clones showed either a
reduced amount of CSN5 RNA in the zygotic pattern
(Fig. 1F) or no detectable
CSN5 RNA (not shown).
Flies carrying CSN5 germline clones laid eggs with a range of abnormal phenotypes that were affected by temperature (Fig. 2, Table 1). Flies grown at 25°C laid eggs with phenotypes closest to normal. The most frequent defects at 18°C were different from those at 29°C. At 18°C many of the defective eggs had fused dorsal appendages (Fig. 2B). At 29°C there was an increasing frequency of properly separated but short dorsal appendages (Fig. 2E). These results suggest that aberrations in patterning the follicular epithelium predominate at 18°C, while defects in follicle cell migration predominate at 29°C.
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Because the eggshell phenotypes were only partially penetrant, it was
possible that they were caused by somatic, rather than germline, CSN5
clones. To test this possibility, we induced somatic clones in the ovary by
using the follicle cell driver E22c-GAL4 to induce expression of
UAS-FLP (Duffy et al.,
1998). Under these conditions, there were no eggshell defects at
any temperature, indicating that this requirement for CSN5 function
is limited to the germline.
In addition to the eggshell defects, the viability of CSN5 mutants also depends on temperature. At 29°C the original P-element mutation is lethal during early development with fewer than 1% of the mutant larvae becoming prepupae. By contrast, at 18°C 90% of the mutant larvae pupariate and 1-2% escape as adults. Mobilization of the original P-element insertion confirmed that it was responsible, not only for lethality, but also for the eggshell defects; precise excisions were viable and had normal dorsal appendages.
Maternal expression of CSN5 is required for embryonic
dorsal-ventral patterning
Some mutations that disrupt the DV patterning of the eggshell also affect
the patterning of the embryo. To look for effects on the embryonic DV fate
map, we used as markers the expression of three zygotic genes:
decapentaplegic (dpp), rhomboid (rho) and
twist (twi) (Fig.
3). dpp is expressed on the dorsal side of the embryo as
well as its anterior and posterior ends
(St Johnston and Gelbart,
1987). rho is expressed in two, eight-cell-wide
ventrolateral domains and later also in a narrow stripe on the dorsal side of
the embryo (Bier et al., 1990
).
twi, a marker for the mesoderm, is expressed ventrally in the embryo
(Thisse et al., 1988
).
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For all three of the markers, many of the CSN5-mutant embryos appeared to be ventralized (Fig. 3B,E,H). In these embryos dpp expression on the dorsal side was reduced or absent. The dorsal rho stripe was reduced and the lateral stripes were moved dorsally. twi expression appeared to expand dorsally about halfway around the embryo. Some embryos showed stronger ventralization at their anterior or posterior ends (data not shown). There were also infrequent embryos that appeared to be dorsalized (Fig. 3C,F,I).
CSN5 is also required for anterior-posterior
polarization
To characterize CSN5 mutants further, we examined the spatial
localization of the RNAs for two determinants of AP polarity, bicoid
(bcd) and oskar (osk). The localization of
bcd RNA to the anterior pole of the oocyte is crucial in the
establishment of AP polarity
(Nusslein-Volhard et al.,
1987; Berleth et al.,
1988
; St Johnston et al.,
1989
). In CSN5 mutant oocytes and embryos, bcd
mRNA was abnormally expressed in 10-15% of oocytes
(Fig. 4). In these abnormal
oocytes, the bcd mRNA is diffusely distributed and sometimes
accumulated near the center of the oocyte
(Fig. 4B). In mutant embryos,
the bcd RNA often shifted toward the dorsal side of the embryo
(Fig. 4F).
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The posterior pole of the egg chamber is defined by the tight, posterior
localization of osk RNA (Ephrussi
et al., 1991; Kim-Ha et al.,
1991
). Although most CSN5-mutant oocytes and embryos were
nearly normal, osk RNA in 10-15% of mutant oocytes and embryos was
reduced or mislocalized (Fig.
4). In the abnormal oocytes, the osk RNA was typically
diffuse or concentrated in the center of the oocyte
(Fig. 4D). Only small amounts
were localized at the posterior pole. In the abnormal embryos only a small
amount of osk RNA at the posterior pole remained. In these embryos
the osk RNA appeared to be shifted slightly dorsally from its normal
position at the extreme posterior end (Fig.
4H).
Since the localization of osk and bcd RNAs depends on
polarization of the microtubule lattice, we used a reporter for the motor
protein kinesin to examine microtubule organization in CSN5 germline
clones (Clark et al., 1994).
Kinesin moves toward the plus ends of microtubules, and in stage 8-9 wild-type
egg chambers kinesin-ß-gal localizes to the posterior of the oocyte.
However, in some CSN5-mutant oocytes kinesin-ß-gal staining was
diffuse or mislocalized (not shown).
CSN5 may also be required for proper pole cell
organization
In addition to its role in determining the AP axis, CSN5 may have
a distinct role in pole cell development. In normal embryos, the pole cells
form as a tight, contiguous cluster at the posterior end of the embryo
(Fig. 4I). As gastrulation and
germ band extension begin, somatic epithelial cells at the posterior end of
the embryo form a shallow cup that will eventually become the posterior midgut
invagination. The pole cells adhere to this cup and remain tightly clustered
on its surface as they are conveyed over the dorsal side of the embryo and
then into its interior. In CSN5-mutant embryos the number of pole
cells is often reduced, as might be expected because of the inefficient
localization of oskar RNA (Fig.
4J). In addition, the pole cells are occasionally found in a
loose, non-contiguous group near, but not tightly associated with, the
posterior end of the embryo (Fig.
4K). This is an unusual phenotype, not seen in other mutants that
impair the formation of pole plasm. Thus, in addition to its role in
oskar RNA localization, CSN5 may have a separate role in
organizing the pole cell cluster.
CSN5 is required for grk signaling
Since CSN5 germline clones caused defects in both the AP and DV
axes, it seemed possible that grk signaling was compromised
(Gonzalez-Reyes et al., 1995;
Roth et al., 1995
). As
described in the introduction, grk is unusual among axis-determining
genes in being required for both axes.
To assess the role of CSN5 in grk signaling, we used
reporters for either the posterior or the dorsal Grk signal. In the absence of
the posterior Grk signal, the posterior follicle cells appear to adopt the
anterior follicle cell fate and express markers that are characteristic of the
border cells (Gonzalez-Reyes et al.,
1995; Roth et al.,
1995
). We used two such markers, an enhancer trap called PZ6356
(Fig. 5A) and a
slbo-lacZ enhancer trap (Fig.
5C) (Montell et al.,
1992
; Tinker et al.,
1998
; Liu and Montell,
1999
), to monitor whether CSN5 is required for the early
Grk signal. For both markers, loss of CSN5 from the germline caused
lacZ expression in the posterior follicle cells of many egg chambers,
suggesting a reduction in Grk signaling
(Fig. 5B,D). To monitor
EGFR signaling to the dorsal follicle cells at stages 9 and 10, we
used a kekkon (kek)-lacZ reporter construct
(Fig. 5E). Because the
kek gene acts downstream of the EGFR pathway in the follicle
cells, it can serve as a sensitive indicator of grk activity coming
from the oocyte (Musacchio and Perrimon,
1996
; Sapir et al.,
1998
). We found that at 18°C kek expression is
abnormal in about a third of CSN5-mutant egg chambers at stage 10
(but only 3-4% at 25°C). In most of these egg chambers, expression in the
dorsal anterior follicle cells over the oocyte was reduced or, rarely, absent
(Fig. 5F,G). A small number of
egg chambers show broader expression of kek in the follicle cells,
probably reflecting the small number of dorsalized embryos arising from these
mutant egg chambers (Fig. 5H).
We conclude that in most egg chambers both posterior and dorsal Grk signaling
are impaired in CSN5-mutant germline clones.
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Further evidence that CSN5 affects Grk signaling came from testing for genetic interactions between CSN5 and either grk or EGFR. Females heterozygous for strong grk alleles lay eggs with fused or partially fused dorsal appendages (Table 2). This dominant phenotype provides a sensitive background for detecting interactions. With the exception of a precise P-element excision, all CSN5 alleles showed strong enhancement of the dominant grk phenotype (Table 2). In addition, CSN5L4032 weakly enhanced the dominant eggshell phenotype of a loss of function EGFR allele, EGFRf2.
|
These results suggested that production of grk RNA or protein might be affected in CSN5 germline clones. In situ hybridization using a grk probe showed normal or nearly normal localization of grk RNA in most CSN5-mutant stage 10 oocytes (Fig. 6B). In some of these mutant oocytes the messenger was improperly localized, probably because the oocyte nucleus was no longer located at the dorsal corner of the oocyte (Fig. 6C). Interestingly, in these oocytes the `dorsal' follicle cells were often columnar as though the nucleus had been properly localized at an earlier stage (Fig. 6C). A northern blot showed nearly normal amounts of grk mRNA in ovaries carrying CSN5-mutant germline clones, consistent with the strong signals seen by in situ hybridization in most oocytes (Fig. 6G).
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Immunostaining of egg chambers using anti-Grk antibodies showed a more extreme effect. Grk protein was strongly reduced in CSN5 mutants compared with controls, although the residual protein usually appeared to be properly localized (Fig. 6D-F). This reduction was confirmed by western blot analysis (Fig. 6H). There were also a few cases of Grk protein mislocalization, sometimes being present all along the anterior end of the oocyte (data not shown).
The reduction in Grk protein appeared to be most extreme at early stages in oogenesis. Grk expression begins in region 2a in wild-type germaria. The signal appears in several cells per cyst in regions 2a and 2b and then becomes concentrated in the oocyte cytoplasm by region 3 (Fig. 7A). In viable, hypomorphic combinations such as CSN5ex21/CSN5L4032, we could not detect Grk expression in the germarium (Fig. 7B). With this combination Grk does become detectable from stage 2-3 onwards (data not shown), suggesting that a reduction in CSN5 causes a delay in the beginning of Grk accumulation (see Discussion). Taken together these results show that the major effect of CSN5 mutations appears to be on grk RNA translation or on stability of the protein.
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CSN5 mutations activate a meiotic checkpoint
Because of the similarity between the CSN5 and
spindle-class phenotypes, we tested for a connection between
CSN5 and the meiotic checkpoint mediated by mei-41. As
mentioned above, the viable hypomorphic combination
CSN5ex21/CSN5L4032 caused a reduction in Grk
protein level, especially during the early stages of oogenesis
(Fig. 7B). Five to fifteen
percent of eggs laid by these transheterozygotes had fused dorsal appendages,
indicating a partial reduction of Grk. When
CSN5ex21/CSN5L4032 flies were also
homozygous-mutant for mei-41, however, the normal Grk protein level
was restored (Fig. 7C), and the
eggshell phenotype was rescued (not shown).
Interestingly, checkpoint activation leads to modification of the Vasa
protein, as shown by a slightly reduced mobility during SDS polyacrylamide gel
electrophoresis (Ghabrial and Schupbach,
1999). This result is relevant to the spindle-class and
CSN5 phenotypes because Vasa regulates translation of Gurken and, as
a consequence, axial patterning (Styhler
et al., 1998
; Tomancak et al.,
1998
). This Vasa modification is checkpoint dependent since it is
present in spn-B mutants but absent in mei-41 spn-B double
mutants (Ghabrial and Schupbach,
1999
).
We detected a similar reduced mobility of Vasa protein in CSN5 mutants (Fig. 7D). For viable CSN5 mutants there were two Vasa bands: one corresponding to Vasa from wild-type ovaries and a second with lower mobility. In stronger mutant combinations, most of the Vasa protein was modified, while in weaker combinations most Vasa had normal mobility. The shift in Vasa mobility was suppressed by mei-41 mutations. Interestingly, removal of one dose of mei-41 completely restored normal Vasa mobility for a weak CSN5 combination. For stronger CSN5 mutants, full restoration of Vasa mobility required removal of both mei-41 genes (Fig. 7E).
The gene mei-W68 is required for the initiation of meiotic
recombination in Drosophila ovaries and is likely to induce DNA
double strand breaks (DSBs) as recombination begins
(McKim and Hayashi-Hagihara,
1998). Mutations in mei-W68 were shown to rescue
spindle-class defects, including Grk protein accumulation, eggshell
morphology and Vasa modification (Ghabrial
and Schupbach, 1999
). These results suggested that since DSBs were
not formed in the absence of mei-W68, DNA repair by the
spindle-class genes was not required. A similar interaction was seen
between mei-W68 and CSN5. Hetrerozygosity for
mei-W68 was sufficient to suppress the phenotypes of both strong and
weak CSN5 allelic combinations
(Fig. 7E).
These data demonstrate that absence of CSN5 function during meiosis activates a DNA-damage checkpoint that is mediated by Mei-41. Because the reduction in DSBs in mei-W68 heterozygotes removes the requirement for CSN5, it is likely that CSN5 promotes DNA repair, as do the spindle-class genes.
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DISCUSSION |
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CSN5 mutations activate a mei-41-dependant meiotic
checkpoint
Previous studies have shown that the accumulation of Grk protein can be
affected by activation of a meiotic checkpoint in response to the persistence
of DNA double-strand breaks (Ghabrial and
Schupbach, 1999). Mutations in several genes that play a role in
DNA repair (okra, spn-B, spn-C and spn-D) activate this
meiotic checkpoint and disrupt axial patterning in the oocyte. There is a
remarkable similarity between the CSN5-mutant phenotype and defects
caused by mutations in these spindle-class genes (described by
Gonzalez-Reyes et al., 1997
;
Ghabrial et al., 1998
;
Ghabrial and Schupbach, 1999
).
In both cases mutant females produced eggs with a variety of partially
penetrant eggshell defects: mild or strongly ventralized, dorsalized, or small
eggs or eggs with multiple dorsal appendages. Embryonic patterning was also
disrupted, and both axes were affected. As had been seen in
spindle-class mutants, the oocyte of some CSN5-mutant egg
chambers was positioned laterally or at the anterior end, and some had defects
in karyosome morphology (data not shown). There was also a similar, strong
reduction in Grk protein, with one intriguing difference. At early stages of
oogenesis in CSN5 mutants, the level of Grk protein was always
strongly reduced, both in germline clones of the strong
CSN5L4032 allele and in hypomorphic combinations of
CSN5L4032 with viable excision mutants
(Fig. 7). Although Grk was also
strongly reduced in CSN5L4032 germline clones at later
stages (Fig. 6), it often
appeared to be present at higher levels than in the germarium. With the
hypomorphic combinations, it was often difficult to detect any reduction in
Grk protein at later stages. By contrast, in spn-B and spn-D
mutants, Grk accumulates normally in early oogenesis but then declines and is
often undetectable by stage 9-10 (Ghabrial
et al., 1998
). In okr mutants, the amount of Grk protein
varies from one egg chamber to the next in a single ovariole, but a bias
towards lower levels at early stages was not reported
(Ghabrial et al., 1998
). Thus,
there seem to be three different patterns of Grk accumulation in these
mutants. CSN5 mutants appear to cause a more immediate response of
Grk to DNA damage than do spn-B and spn-D mutants.
Because of the similarities between the phenotypes and because at least two
of the spindle-class genes, okr and spn-B, encode
components of the RAD52 DNA repair pathway, it seems likely that CSN5
directly or indirectly regulates DSB repair. The fact that mei-41 and
mei-W68 mutations can suppress the CSN5 phenotypes
reinforces this conclusion. Kinases in the ATM/ATR subfamily that includes
Mei-41 play a central role in checkpoint-mediated responses to DNA damage (for
reviews, see Melo and Toczysky, 2002;
Weinert, 1998;
Murakami and Nurse, 2000
).
These checkpoint kinases are thought to act as sensors of DNA damage, becoming
activated on binding damaged DNA. Phosphorylation of several downstream
effectors, including the Chk1 and Chk2 kinases and p53, then restrains cell
cycle progression until the DNA damage is repaired and the checkpoint kinases
dissociate from the DNA. In Drosophila mei-41 mutants, the checkpoint
cannot be activated, and oocytes with damaged DNA, such as those mutant for
spindle-class genes, can proceed through oogenesis. Suppression of
CSN5 phenotypes by mei-41 mutations demonstrates that the
CSN5-mutant lesion acts upstream of the DNA damage checkpoint and
suggests that DSBs arising during meiotic recombination cannot be efficiently
repaired in CSN5-mutant cells
(Fig. 7).
Suppression by mei-W68 restricts the possible role of CSN5
further. mei-W68 encodes a topoisomerase II-like protein homologous
to S. cerevisiae Spo11 and has been proposed to create the DSBs
needed to initiate meiotic recombination
(McKim and Hayashi-Hagihara,
1998). In flies mutant for mei-W68, DSBs are absent and
meiotic recombination is eliminated. In double mutants of mei-W68
with either okr, spn-B or spn-C, Grk protein accumulation
and eggshell patterning are normal and other spindle-class defects
are suppressed (Ghabrial and Schupbach,
1999
). We found that heterozygosity for mei-W68 was
sufficient to suppress hypomorphic CSN5-mutant phenotypes
(Fig. 7). Combination of this
result with the mei-41 suppression result indicates that CSN5 acts in
the recombination pathway to regulate the formation of DSBs or their
successful repair (Fig. 8).
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vasa mutants show similar effects on axis determination and Grk
protein accumulation as do spindle mutants and CSN5 GLCs
(Styhler et al., 1998).
However, the vasa phenotypes are not suppressed by mei-41 or
mei-W68 mutations, indicating that Vasa acts downstream of the
meiotic checkpoint (Ghabrial and
Schupbach, 1999
). Indeed, Vasa is one of the targets of Mei-41
activity as Vasa electrophoretic mobility is changed in spn-B mutants
but restored in mei-41 spn-B double mutants
(Ghabrial and Schupbach,
1999
). Since Vasa protein binds to grk mRNA and is
required for both its localization in the oocyte and its translation, it seems
likely that the checkpoint effects on Grk accumulation are directly mediated
by Vasa, although other Mei-41 targets cannot be excluded
(Fig. 8). Our results that show
effects of CSN5 mutants on Vasa mobility are entirely consistent with
the previous spn-B results, as would be expected if both types of
mutants activate the same checkpoint.
We propose that in CSN5-mutant oocytes DSBs created by Mei-W68 during meiotic recombination are repaired more slowly than in wild type. Accumulation of unrepaired DNA breaks would then activate the mei-41-dependent checkpoint leading to a block in the progression of meiotic prophase (Fig. 8). Since activated Mei-41 is an ATR-related kinase, it might modify Vasa directly or through downstream kinases such as Chk1 or Chk2. Modified Vasa would then prevent efficient Grk translation. Because CSN5 mutants are likely to affect the stability rather than the presence or absence of repair proteins, the DNA DSBs might be slowly repaired during the checkpoint-induced delay, thereby allowing cell cycle progression to resume. Delayed repair might explain why the early CSN5 effects on Grk expression are stronger than at later times. It might also explain why CSN5-mutant phenotypes are weaker and less penetrant than in okra and spn-B mutants, in which repair proteins are absent and DNA probably remains unrepaired.
CSN5 and DNA repair
How might CSN5 regulate DNA repair? Two mechanisms of CSN activity have
been reported, and either might affect the activity or stability of proteins
involved in DNA repair. In addition, since there is an excess of CSN5 relative
to other CSN subunits in many cells (Yang
et al., 2002), CSN5 might regulate DNA repair independent of the
large CSN complex.
The best-documented mechanism for CSN activity works through regulation of
the SCF (Skp1/cullin-1/F-box) ubiquitin ligases
(Lyapina et al., 2001;
Yang et al., 2002
). This
pathway is attractive here because SCF-dependent ubiquitination mediates the
degradation of many cell-cycle regulators, including not only
p27kip1, but also cyclins E, A and B, CDK inhibitor p21, E2F1,
ß-catenin and I
B
(Michel and Xiong, 1998
;
Russell et al., 1999
;
Yu et al., 1998
;
Carrano et al., 1999
;
Marti et al., 1999
;
Kitagawa et al., 1999
;
Hatakeyama et al., 1999
).
Recently, a connection has been made in C. elegans between the SCF
complex and the regulation of meiosis. Members of the Skp1-related
(skr) gene family in C. elegans are required for the
restraint of cell proliferation, progression through the pachytene stage of
meiosis, and formation of bivalent chromosomes at diakinesis
(Nayak et al., 2002
).
The CSN regulates SCF activity by removing the ubiquitinlike protein Nedd8
from the cullin subunit of SCF (Lyapina et
al., 2001). Nedd8/Rub1 is covalently attached to target proteins
through an enzymatic cascade analogous to ubiquitination
(Lammer et al., 1998
;
Liakopoulos et al., 1998
;
Osaka et al., 2000
). It is
ligated to all cullin family proteins, and so far cullins are the only known
targets for neddylation (Hori et al.,
1999
; Read et al.,
2000
). Nedd8 modification enhances the ubiquitinating activity of
the SCF complex in vitro and is required in vivo for embryogenesis in both
mice and nematodes (Kawakami et al.,
2001
; Tateishi et al.,
2001
; Jones et al.,
2002
). As the CSN mediates cleavage of the Nedd8 conjugate, it can
antagonize SCF-dependent protein degradation. For example CSN inhibits
ubiquitination and degradation of p27kip1 in vitro and injection of
the purified complex inhibited the G1-S transition in cultured cells
(Yang et al., 2002
).
Although this deneddylation activity of the CSN would explain our results,
the kinase activity associated with the CSN might also be important. This
kinase activity co-purifies with the CSN complex though it is uncertain
whether it is intrinsic to one of the CSN subunits
(Bech-Otschir et al., 2002). It
phosphorylates and stabilizes the Jun transcription factor against proteasomal
degradation (Musti et al.,
1997
). Conversely, it sensitizes p53 degradation by the
SCF-ubiquitin pathway (Bech-Otschir et al.,
2001
).
Although the DNA repair-related targets of CSN5 or the CSN remain unclear, proteins encoded by the spindle-class genes or by mei-W68 are strong candidates (see Fig. 8). The deneddylation activity of the CSN might protect a DNA repair protein from SCF-dependent degradation. Alternatively, the kinase activity might promote Mei-W68 turnover, thereby limiting the production of DSBs. Further investigation may help to distinguish among these and other hypotheses and find direct targets for CSN5 in oogenesis.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Bech-Otschir, D., Kraft, R., Huang, X., Henklein, P., Kapelari,
B., Pollmann, C. and Dubiel, W. (2001). COP9
signalosome-specific phosphorylation targets p53 to degradation by the
ubiquitin system. EMBO J.
20,1630
-1639.
Bech-Otschir, D., Seeger, M. and Dubiel, W.
(2002). The COP9 signalosome: at the interface between signal
transduction and ubiquitin-dependent proteolysis. J. Cell
Sci. 115,467
-473.
Berleth, T., Burri, M., Thoma, G., Bopp, D., Richstein, S., Frigerio, G., Noll, M. and Nusslein-Volhard, C. (1988). The role of localization of bicoid RNA in organizing the anterior pattern of the Drosophila embryo. EMBO J. 7,1749 -1756.[Abstract]
Bier, E., Jan, L. Y. and Jan, Y. N. (1990). rhomboid, a gene required for dorsoventral axis establishment and peripheral nervous system development in Drosophila melanogaster.Genes. Dev. 4,190 -203.[Abstract]
Cant, K., Knowles, B. A., Mooseker, M. S. and Cooley, L. (1994). Drosophila singed, a Fascin homolog, is required for actin bundle formation during oogenesis and bristle extension. J. Cell Biol. 125,369 -380.[Abstract]
Carrano, A. C., Eytan, E., Hershko, A. and Pagano, M. (1999). SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27. Nat. Cell Biol. 1, 193-199.[CrossRef][Medline]
Chou, T. B. and Perrimon, N. (1992). Use of a
yeast site-specific recombinase to produce female germline chimeras in
Drosophila. Genetics
131,643
-653.
Chou, T. B. and Perrimon, N. (1996). The
autosomal FLP-DFS technique for generating germline mosaics in Drosophila
melanogaster. Genetics 144,1673
-1679.
Clark, I., Giniger, E., Ruohola-Baker, H., Jan, L. Y. and Jan, Y. N. (1994) Transient posterior localization of a kinesin fusion protein reflects anteroposterior polarity of the Drosophila oocyte. Curr. Biol. 4,289 -300.[Medline]
Duffy, J. B., Harrison, D. A. and Perrimon, N.
(1998). Identifying loci required for follicular patterning using
directed mosaics. Development
125,2263
-2271.
Engels, W. R., Benz, W. K., Preston, C. R., Graham, P. L.,
Phillis, R. W. and Robertson, H. M. (1987). Somatic effects
of P element activity in Drosophila melanogaster: pupal lethality.
Genetics 117,745
-757.
Ephrussi, A., Dickinson, L. K. and Lehmann, R. (1991). Oskar organizes the germ plasm and directs localization of the posterior determinant nanos. Cell 66, 37-50.[Medline]
Freilich, S., Oron, E., Kapp, Y., Nevo-Caspi, Y., Orgad, S., Segal, D. and Chamovitz, D. A. (1999). The COP9 signalosome is essential for development of Drosophila melanogaster. Curr. Biol. 9,1187 -1190.[CrossRef][Medline]
Ghabrial, A. and Schupbach, T. (1999) Activation of a meiotic checkpoint regulates translation of Gurken during Drosophila oogenesis. Nat. Cell Biol. 1, 354-357.[CrossRef][Medline]
Ghabrial, A., Ray, R. P. and Schupbach, T.
(1998) okra and spindle-B encode components of
the RAD52 DNA repair pathway and affect meiosis and patterning in
Drosophila oogenesis. Genes Dev.
12,2711
-2723.
Glickman, M. H., Rubin, D. M., Coux, O., Wefes, I., Pfeifer, G., Cjeka, Z., Baumeister, W., Fried, V. A. and Finley, D. (1998). A subcomplex of the proteasome regulatory particle required for ubiquitin-conjugate degradation and related to the COP9-signalosome and eIF3. Cell 94,615 -623.[Medline]
Gonzalez-Reyes, A. and St Johnston, D. (1998).
The Drosophila AP axis is polarised by the cadherin-mediated
positioning of the oocyte. Development
125,3635
-3644.
Gonzalez-Reyes, A., Elliott, H. and St Johnston, D. (1995). Polarization of both major body axes in Drosophila by gurken-torpedo signalling. Nature 375,654 -658.[CrossRef][Medline]
Gonzalez-Reyes, A., Elliott, H. and St Johnston, D.
(1997). Oocyte determination and the origin of polarity in
Drosophila: the role of the spindle genes.
Development 124,4927
-4937.
Harland, R. M. (1991). In situ hybridization: an improved whole-mount method for Xenopus embryos. Methods Cell Biol. 36,685 -695.[Medline]
Hatakeyama, S., Kitagawa, M., Nakayama, K., Shirane, M.,
Matsumoto, M., Hattori, K., Higashi, H., Nakano, H., Okumura, K., Onoe, K.,
Good, R. A. and Nakayama, K. (1999) Ubiquitin-dependent
degradation of IkappaBalpha is mediated by a ubiquitin ligase Skp1/Cul 1/F-box
protein FWD1. Proc. Natl. Acad. Sci. USA
96,3859
-3863.
Hori, T., Osaka, F., Chiba, T., Miyamoto, C., Okabayashi, K., Shimbara, N., Kato, S. and Tanaka, K. (1999). Covalent modification of all members of human cullin family proteins by NEDD8. Oncogene 18,6829 -6834.[CrossRef][Medline]
Huynh, J. R. and St Johnston, D. (2000). The
role of BicD, Egl, Orb and the microtubules in the restriction of meiosis to
the Drosophila oocyte. Development
127,2785
-2794.
Jones, D., Crowe, E., Stevens, T. A. and Candido, E. P. (2002). Functional and phylogenetic analysis of the ubiquitylation system in Caenorhabditis elegans: ubiquitin-conjugating enzymes, ubiquitin-activating enzymes, and ubiquitin-like proteins. Genome Biol. 3,0002.1 -0002.15.
Kawakami, T., Chiba, T., Suzuki, T., Iwai, K., Yamanaka, K.,
Minato, N., Suzuki, H., Shimbara, N., Hidaka, Y., Osaka, F., Omata, M. and
Tanaka, K. (2001). NEDD8 recruits E2-ubiquitin to SCF E3
ligase. EMBO J. 20,4003
-4012.
Kim, T., Hofmann, K., von Arnim, A. G. and Chamovitz, D. A. (2001) PCI complexes: pretty complex interactions in diverse signaling pathways. Trends Plant Sci. 6, 379-386.[CrossRef][Medline]
Kim-Ha, J., Smith, J. L. and Macdonald, P. M. (1991). oskar mRNA is localized to the posterior pole of the Drosophila oocyte. Cell 66, 23-35.[Medline]
Kitagawa, M., Hatakeyama, S., Shirane, M., Matsumoto, M.,
Ishida, N., Hattori, K., Nakamichi, I., Kikuchi, A., Nakayama, K.-I. and
Nakayama, K. (1999). An F-box protein, FWD1, mediates
ubiquitindependent proteolysis of beta-catenin. EMBO
J. 18,2401
-2410.
Kooistra, R., Vreeken, K., Zonneveld, J. B., de Jong, A., Eeken, J. C., Osgood, C. J., Buerstedde, J. M., Lohman, P. H. and Pastink, A. (1997). The Drosophila melanogaster RAD54 homolog, DmRAD54, is involved in the repair of radiation damage and recombination. Mol Cell Biol. 17,6097 -6104.[Abstract]
Lammer, D., Mathias, N., Laplaza, J. M., Jiang, W., Liu, Y.,
Callis, J., Goebl, M. and Estelle, M. (1998). Modification of
yeast Cdc53p by the ubiquitin-related protein rub 1p affects function of the
SCFCdc4 complex. Genes Dev.
12,914
-926.
Liakopoulos, D., Doenges, G., Matuschewski, K. and Jentsch,
S. (1998). A novel protein modification pathway related to
the ubiquitin system. EMBO J.
17,2208
-2214.
Liu, Y. and Montell, D. J. (1999).
Identification of mutations that cause cell migration defects in mosaic
clones. Development 126,1869
-1878.
Lyapina, S., Cope, G., Shevchenko, A., Serino, G., Tsuge, T.,
Zhou, C., Wolf, D. A., Wei, N., Shevchenko, A. and Deshaies, R. J.
(2001). Promotion of NEDD-CUL1 conjugate cleavage by COP9
signalosome. Science
292,1382
-1385.
Marti, A., Wirbelauer, C., Scheffner, M. and Krek, W. (1999). Interaction between ubiquitin-protein ligase SCFSKP2 and E2F-1 underlies the regulation of E2F-1 degradation. Nat. Cell Biol. 1,14 -19.[CrossRef][Medline]
McKim, K. S. and Hayashi-Hagihara, A. (1998)
mei-W68 in Drosophila melanogaster encodes a Spoll homolog: evidence that the
mechanism for initiating meiotic recombination is conserved. Genes
Dev. 12,2932
-2942.
Melo, J. and Toczyski, D. (2002). A unified view of the DNA-damage checkpoint. Curr. Opin. Cell Biol. 14,237 -245.[CrossRef][Medline]
Michel, J. and Xiong, Y. (1998). Human CUL-1, but not other cullin family members, selectively interacts with SKP1 to form a complex with SKP2 and cyclin A. Cell Growth Differ. 9, 435-449.[Abstract]
Montell, D. J., Rorth, P. and Spradling, A. C. (1992). slow border cells, a locus required for a developmentally regulated cell migration during oogenesis, encodes Drosophila C/EBP. Cell 71,51 -62.[Medline]
Murakami, H. and Nurse, P. (2000). DNA replication and damage checkpoints and meiotic cell cycle controls in the fission and budding yeasts. Biochem. J. 349, 1-12.
Musacchio, M. and Perrimon, N. (1996). The Drosophila kekkon genes: novel members of both the leucine-rich repeat and immunoglobulin superfamilies expressed in the CNS. Dev. Biol. 178,63 -76.[CrossRef][Medline]
Musti, A. M., Treier, M. and Bohmann, D.
(1997). Reduced ubiquitindependent degradation of c-Jun after
phosphorylation by MAP kinases. Science
275,400
-402.
Nayak, S., Santiago, F., Jin, H., Lin, D., Schedl, T. and Kipreos, E. (2002). The Caenorhabditis elegans Skp1-related gene family; diverse functions in cell proliferation, morphogenesis, and meiosis. Curr. Biol. 4,277 -287.[CrossRef]
Neuman-Silberberg, F. S. and Schupbach, T. (1993). The Drosophila dorsoventral patterning gene gurken produces a dorsally localized RNA and encodes a TGF alpha-like protein. Cell 75,165 -174.[Medline]
Neuman-Silberberg, F. S. and Schupbach, T.
(1994). Dorsoventral axis formation in Drosophila
depends on the correct dosage of the gene gurken.Development 120,2457
-2463.
Nusslein-Volhard, C., Frohnhofer, H. G. and Lehmann, R. (1987). Determination of anteroposterior polarity in Drosophila. Science 238,1675 -1681.[Medline]
Osaka, F., Saeki, M., Katayama, S., Aida, N., Toh-E, A.,
Kominami, K., Toda, T., Suzuki, T., Chiba, T., Tanaka, K. and Kato, S.
(2000). Covalent modifier NEDD8 is essential for SCF
ubiquitin-ligase in fission yeast. EMBO J.
19,3475
-3484.
Read, M. A., Brownell, J. E., Gladysheva, T. B., Hottelet, M.,
Parent, L. A., Coggins, M. B., Pierce, J. W., Podust, V. N., Luo, R. S., Chau,
V. and Palombella, V. J. (2000). Nedd8 modification of cul-1
activates SCF(beta(TrCP))-dependent ubiquitination of IkappaBalpha.
Mol. Cell Biol. 20,2326
-2333.
Roeder, G. S. (1997). Meiotic chromosomes: it
takes two to tango. Genes Dev.
11,2600
-2621.
Roth, S., Neuman-Silberberg, F. S., Barcelo, G. and Schupbach, T. (1995). cornichon and the EGF receptor signaling process are necessary for both anterior-posterior and dorsal-ventral pattern formation in Drosophila. Cell 81,967 -978.[Medline]
Russell, A., Thompson, M. A., Hendley, J., Trute, L., Armes, J. and Germain, D. (1999). Cyclin D1 and D3 associate with the SCF complex and are coordinately elevated in breast cancer. Oncogene 18,1983 -1991[CrossRef][Medline]
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory.
Sapir, A., Schweitzer, R. and Shilo, B. Z.
(1998). Sequential activation of the EGF receptor pathway during
Drosophila oogenesis establishes the dorsoventral axis.
Development 125,191
-200.
Schwechheimer, C. and Deng, X. W. (2001). COP9 signalosome revisited: a novel mediator of protein degradation. Trends Cell Biol. 11,420 -426.[CrossRef][Medline]
Seeger, M., Kraft, R., Ferrell, K., Bech-Otschir, D., Dumdey,
R., Schade, R., Gordon, C., Naumann, M. and Dubiel, W.
(1998). A novel protein complex involved in signal transduction
possessing similarities to 26S proteasome subunits. FASEB
J. 12,469
-478.
Seeger, M., Gordon, C. and Dubiel, W. (2001). Protein stability: the COP9 signalosome gets in on the act. Curr. Biol. 11,643 -646.
Spradling, A. C., Stern, D., Beaton, A., Rhem, E. J., Laverty,
T., Mozden, N., Misra, S. and Rubin, G. M. (1999). The
Berkeley Drosophila genome project gene disruption project. Single P-element
insertions mutating 25% of vital Drosophila genes.
Genetics 153,135
-177.
St Johnston, D. and Gelbart, W. M. (1987). Decapentaplegic transcripts are localized along the dorsal-ventral axis of the Drosophila embryo. EMBO J. 6,2785 -2791.[Abstract]
St Johnston, D., Driever, W., Berleth, T., Richstein, S. and Nusslein-Volhard, C. (1989). Multiple steps in the localization of bicoid RNA to the anterior pole of the Drosophila oocyte. Development 107 Suppl.,13 -19.[Medline]
Styhler, S., Nakamura, A., Swan, A., Suter, B. and Lasko, P.
(1998). vasa is required for GURKEN accumulation in the oocyte,
and is involved in oocyte differentiation and germline cyst development.
Development 125,1569
-1578.
Suh, G. S., Poeck, B., Chouard, T., Oron, E., Segal, D., Chamovitz, D. A. and Zipursky, S. L. (2002). Drosophila JAB1/CSN5 acts in photoreceptor cells to induce glial cells. Neuron 33,35 -46.[Medline]
Tateishi, K., Omata, M., Tanaka, K. and Chiba, T.
(2001). The NEDD8 system is essential for cell cycle progression
and morphogenetic pathway in mice. J. Cell Biol.
155,571
-579.
Tautz, D. and Pfeifle, C. (1989). A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98, 81-85.[Medline]
Thisse, B., Stoetzel, C., Gorostiza-Thisse, C. and Perrin-Schmitt, F. (1988). Sequence of the twist gene and nuclear localization of its protein in endomesodermal cells of early Drosophila embryos. EMBO J. 7,2175 -2183.[Abstract]
Tinker, R., Silver, D. and Montell, D. J. (1998). Requirement for the vasa RNA helicase in gurken mRNA localization. Dev. Biol. 199, 1-10.[CrossRef][Medline]
Tomancak, P., Guichet, A., Zavorszky, P. and Ephrussi, A.
(1998). Oocyte polarity depends on regulation of gurken by Vasa.
Development. 125,1723
-1732.
Tomoda, K., Kubota, Y. and Kato, J. (1999). Degradation of the cyclin-dependent-kinase inhibitor p27Kip1 is instigated by Jab1. Nature 398,160 -165.[CrossRef][Medline]
Verheyen, E. and Cooley, L. (1994). Looking at oogenesis. Methods Cell Biol. 44,545 -561.[Medline]
Wei, N., Tsuge, T., Serino, G., Dohmae, N., Takio, K., Matsui, M. and Deng, X. W. (1998). The COP9 complex is conserved between plants and mammals and is related to the 26S proteasome regulatory complex. Curr. Biol. 8,919 -922.[Medline]
Weinert, T. (1998). DNA damage and checkpoint pathways: molecular anatomy and interactions with repair. Cell 94,555 -558.[Medline]
Yang, X., Menon, S., Lykke-Andersen, K., Tsuge, T., di Xiao, Wang, X., Rodriguez-Suarez, R. J., Zhang, H. and Wei, N. (2002). The COP9 signalosome inhibits p27(kip1) degradation and impedes G1-S phase progression via deneddylation of SCF Cull. Curr. Biol. 12,667 -672.[CrossRef][Medline]
Yu, Z. K., Gervais, J. L. and Zhang, H. (1998).
Human CUL-1 associates with the SKP1/SKP2 complex and regulates p21
(CIP1/WAF1) and cyclin D proteins. Proc. Natl. Acad. Sci.
USA 95,11324
-11329.