|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 11 September 2008
doi: 10.1242/dev.024919
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer
Institute-Frederick, Bldg. 560/22-56, 1050 Boyles Street, Frederick, MD
21702-1201, USA.
2 Whitehead Institute, 9 Cambridge Center, Cambridge, MA 02142, USA.
3 Institute of Molecular and Cell Biology (IMCB), Proteos, 61 Biopolis Drive,
138673 Singapore.
Author for correspondence (e-mail:
kaldis{at}imcb.a-star.edu.sg)
Accepted 20 August 2008
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Cell cycle regulation, Cyclin, Cyclin-dependent kinase (Cdk), Meiosis, Mouse genetics
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). In eukaryotic
cells, several Cdk-cyclin complexes, including Cdk2-cyclin E, Cdk1-cyclin B,
Cdk4-cyclin D, Cdk6-cyclin D and Cdk2-cyclin A, drive cell cycle progression,
and it was believed that their functions are confined to specific stages of
the cell cycle (Morgan, 1997).
For example, Cdk4 and Cdk6 are thought to be involved in early G1, whereas
Cdk2 is essential to complete G1 and initiate S-phase. Cdk4 and Cdk6 form
active complexes with D-type cyclins to initiate the cell division cycle by
phosphorylating the Retinoblastoma protein (Rb). Thereafter, in late G1 phase,
the activation of Cdk2 by cyclin E further phosphorylates Rb and drives cells
through the G1-S restriction point. Later, Cdk2 complexes with cyclin A and is
essential for S-phase progression (for a review, see
Mittnacht, 1998;
Weinberg, 1995). Recently, it
was reported that Cdk2 can also interact with the mitotic cyclin, cyclin B,
but the exact function of this complex is not known
(Aleem et al., 2005). In
addition, it has been reported that Cdk2 is present predominantly in the
nucleus throughout the cell cycle (Moore
et al., 1999; Pines and
Hunter, 1991; Satyanarayana et
al., 2008). Cdk1 (Cdc2a - Mouse Genome Informatics), in
association with cyclin B, is essential to control entry into and exit from
mitosis; Cdk1 is present mainly in the cytoplasm and translocates to the
nucleus only during mitosis after complexing with cyclin B
(Dunphy et al., 1988;
Izumi and Maller, 1993;
Pan et al., 1993;
Riabowol et al., 1989).
In contrast to mammalian cells, in budding yeast a single Cdk, the
transcriptional product of the CDC28 gene, regulates diverse cell
cycle transitions by associating with multiple stage-specific cyclins
(Nasmyth, 1993;
Reed et al., 1982). On the
basis of the concepts derived from the yeast cell cycle, it was hypothesized
that the functions of the multiple Cdks in eukaryotic cells are redundant and
one or two Cdks might be sufficient to drive cells through the different
phases of the cell cycle. In support of this, recent studies have demonstrated
that Cdk2, Cdk4 and Cdk6 single-knockout mice are viable, do
not show any severe phenotypes and display minor defects in cell cycle
properties, indicating functional redundancy between the different Cdks
(Berthet et al., 2003;
Malumbres et al., 2004;
Ortega et al., 2003). Notably,
Cdk1, which was originally identified as an essential mitosis-promoting
kinase, can compensate for the loss of Cdk2 by complexing with cyclin E to
drive cells through the G1-S transition, even though Cdk1 is only 
65%
identical to Cdk2 (Aleem et al.,
2005). In addition, a recent study has demonstrated that Cdk1
alone is sufficient to drive the eukaryotic cell cycle in early embryogenesis
and in mouse embryonic fibroblasts (MEFs)
(Santamaria et al., 2007).
However, at the whole-organism level, the compensation of Cdk2 function by
Cdk1 appears to be only partial, as Cdk2 knockout males and females
are sterile, displaying dysfunctional and atrophic testes and ovaries
(Berthet et al., 2003;
Ortega et al., 2003). This
indicated that Cdk2 is essential for meiosis and that Cdk1 cannot functionally
compensate for the loss of Cdk2. In this context, it is of interest to explore
whether there are any possible ways in which Cdk2 might compensate for the
loss of Cdk1. Deletion of Cdk1 or a gene-trap mutation in the
Cdk1 gene leads to early embryonic lethality (our unpublished
results) (Santamaria et al.,
2007), indicating that Cdk1 is essential for the survival of mice.
This implies that Cdk2 cannot compensate for the loss of Cdk1 when expressed
from its own locus. The inability of Cdk2 to take over the function of Cdk1
could be attributed to: (1) intrinsic differences between the Cdk1 and Cdk2
proteins, such as substrate specificity or interaction with binding partners;
(2) differences in the timing of expression of Cdk1 and Cdk2 during the
different phases of cell cycle; and/or (3) differences in their sub-cellular
localization. It is of interest to explore whether Cdk2 acquires some of the
properties of Cdk1 when Cdk2 is expressed directly from the Cdk1
locus in vivo, and whether it would be able to compensate for the loss of
Cdk1. This hypothesis is derived from recent findings that genetic replacement
of cyclin D1 by cyclin E can rescue the phenotypes of cyclin D1 knockout mice
(Geng et al., 1999).
Similarly, it has been shown that cyclin D2 rescues the loss of cyclin D1 when
expressed from the D1 locus (Carthon et
al., 2005). Furthermore, H-Ras (Hras1) can substitute for K-Ras
(Kras) and supports normal embryonic development
(Potenza et al., 2005). These
studies provide evidence that the timing of expression and the genetic locus
play important roles in determining the functions of a protein. By genetically
replacing Cdk1 with Cdk2, it is possible to study whether Cdk2 can rescue the
loss of Cdk1 in vivo. At the same time, it is of interest to determine whether
Cdk2 can retain its own functions when expressed from the Cdk1 locus.
In this context, it is also important to determine how efficiently Cdk2
performs its own mitotic cell cycle and meiotic functions in germ cells when
expressed from the Cdk1 locus, as Cdk1 cannot functionally rescue the
meiotic functions of Cdk2 in Cdk2-/- mice.
To better understand the importance of genomic location and timing of Cdk2 expression and the possible compensation for loss of Cdk1 by Cdk2, we generated a mouse in which a Cdk2 cDNA was knocked into the Cdk1 locus (Cdk1Cdk2KI). We found that substitution of both copies of Cdk1 by Cdk2 leads to early embryonic lethality, similar to deletion of Cdk1, even though the knockin Cdk2 is expressed from the Cdk1 locus. In addition, in order to study the consequences of Cdk2 expression from the Cdk1 locus on the function of Cdk2, we generated Cdk2-/- Cdk1+/Cdk2KI mice, in which one copy of Cdk2 and one copy of Cdk1 are expressed from the Cdk1 locus with a deletion of the Cdk2 gene in the original Cdk2 locus. From this study, we found that both male and female Cdk2-/- Cdk1+/Cdk2KI mice are sterile, similar to Cdk2-/- mice, even though they express the Cdk2 protein from the Cdk1 locus in testis.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
),
we first inserted an FRT site into intron 2 of Cdk1. Then, a cassette
harboring [Cdk1 homology arm 5'-Cdk2
cDNA-IRES-β-galactosidase-FRT-loxP-PGK-EM7-neomycin-poly(A)-FRT-loxP
Cdk1 homology arm 3'] was inserted in place of exon 3 of the
Cdk1 locus. The insertion site maintains all Cdk1 exon 3
splicing sequences and results in a transcript including Cdk1 exon 1
(5' UTR), Cdk1 exon 2 (including the ATG start codon plus 11
amino acids) and the Cdk2
12-HA cDNA. We
designed this construct to induce Cdk212AACdk1 expression under
potential regulatory sequences including the Cdk1 5' UTR,
promoter, intron 1 and intron 2. Moreover, over the first 12 amino acids, Cdk1
and Cdk2 are very similar [they differ by four amino acids (in red in
Fig. 1A)], suggesting that this
region would not affect Cdk2 properties (see Fig. S1 in the supplementary
material). The Cdk1Cdk2KI locus was then retrieved into
pBluescriptLight-HSVTK (Liu et al.,
2003) and, after recombination, a 22 kb fragment of the
Cdk1Cdk2KI locus was selected by ampicillin and kanamycin
resistance. After electroporation and selection with G418 and gancyclovir,
three independent embryonic stem cell clones were identified which had the
Cdk1Cdk2KI locus correctly targeted. Positive clones were
screened by β-galactosidase expression, Southern blot and PCR. The
following primers were used: 5'-ACCATGTATATGTTAGATCGTAG-3'
(PKO553), 5'-TCGCTTTCAAGTCTGATCTTCT-3' (PKO554) and
5'-CGATATTAGGGTGATTAAGTTCC-3' (PKO043). Wild-type clones yield a
band of 300 bp, whereas the mutant clone produces a band of 450 bp. Germline
transmission was obtained from two clones and these were used to generate
chimeric animals.
Mice and surgical procedures
Mice were housed under standard conditions and were maintained on a 12-hour
light/dark cycle. Mice were fed a standard chow diet containing 6% crude fat
and were treated in compliance with the National Institutes of Health
guidelines for animal care and use.
Twelve- to fifteen-week-old Cdk2+/+,
Cdk2-/-, Cdk2+/+
Cdk1+/Cdk2KI and Cdk2-/-
Cdk1+/Cdk2KI male mice were used and all animals were operated
upon under sterile conditions between 9 am and 12 pm, as described previously
(Satyanarayana et al., 2003).
Mice were anesthetized by intraperitoneal injection of avertin and were
subjected to partial (70%) hepatectomy (PH). After 2 hours of BrdU labeling,
mice were sacrificed at 24 (n=3), 48 (n=4) and 72
(n=4) hours after PH. For BrdU pulse labeling, 10 µl/g body weight
of labeling reagent (10:1, 5-bromo-2-deoxyuridine:5-fluro-20-deoxyuridine;
Cell Proliferation Kit RPN20, Amersham) was administered intraperitoneally 2
hours before sacrifice. After euthanizing the mice, portions of the liver
lobes were fixed separately for BrdU, Hematoxylin and Eosin and
β-galactosidase staining.
Preparation of mouse embryonic fibroblasts (MEFs) and cell culture
MEFs were prepared as described previously
(Berthet et al., 2003) from
E13.5 Cdk2+/+, Cdk2-/-,
Cdk2+/+ Cdk1+/Cdk2KI and
Cdk2-/- Cdk1+/Cdk2KI embryos. Serum starvation
and stimulation experiments were done as described previously
(Satyanarayana et al.,
2008).
Alamar Blue cell-proliferation assay
Proliferation of Cdk2+/+, Cdk2-/-,
Cdk2+/+ Cdk1+/Cdk2KI and
Cdk2-/- Cdk1+/Cdk2KI MEFs in response to serum
starvation (0.1% FBS) and stimulation (10% FBS) was analyzed in 96-well plates
as described previously (Satyanarayana et
al., 2008).
Immunocytochemistry and confocal microscopy
Serum-starved (DMEM medium with 0.1% FBS, 96 hours) and serum-stimulated
(DMEM medium with 10% FBS) cells from 100-mm culture dishes were transferred
on to coverslips in 12-well plates at a density of 1x105
cells per well and probed with specific antibodies at 0, 6, 12 and 24 hours
after stimulation. Immunocytochemical staining was conducted as described
(Satyanarayana et al., 2008).
Primary antibodies against Cdk2 and HA-tag (to detect knockin Cdk2-HA)
(Berthet et al., 2003) were
used at 1:200 dilution. At each time point, the staining pattern was analyzed
in several low-power fields (63x) and the images were captured with a
confocal laser-scanning microscope (LSM510, Zeiss).
Immunohistochemistry
Slides were rehydrated and a microwave antigen-retrieval step was performed
for 13 minutes in 10 mM sodium citrate (pH 6.0) containing 0.05% Tween 20. The
sections were then treated with 3% hydrogen peroxide for 10 minutes. Blocking
was carried out using 2.5% horse serum, 1% BSA in PBS for 30 minutes. Slides
were incubated at room temperature for 1 hour with the following primary
antibodies: Cdk2 (1:1000), Cdk1 (1:200), HA-Cdk2 (1:5000)
(Berthet et al., 2003) and Cdk2
(Abcam). Antibody detection was achieved using the anti-rabbit ImmPRESS
Reagent Kit (Vector Labs) according to the manufacturer's protocol. Slides
were counterstained with Mayer's Hematoxylin, mounted with Permount mounting
media, and coverslips were applied.
BrdU immunohistochemical staining
BrdU immunohistochemical staining on formalin (Sigma, HT50-1-128)-fixed
5-µm liver sections was performed as described
(Satyanarayana et al., 2008).
An Axioplan2 imaging microscope (Zeiss) was used to photograph and analyze the
BrdU staining pattern (and likewise for H&E, β-galactosidase and
apoptotic staining). At least 3000 nuclei were counted per slide and the
percentage of BrdU-positive nuclei calculated.
Hematoxylin and Eosin (H&E) staining
Frozen sections of liver, testes, ovaries and embryos were warmed to room
temperature for 20 minutes. The tissue sections were fixed in acetone for
10 minutes and then air dried. Slides were rinsed with distilled water (2
minutes), incubated in Hematoxylin (Richard-Allan Scientific, 7231) for 3
minutes, and then washed with distilled water twice for 2 minutes. The slides
were treated with clarifier (Richard-Allan Scientific, 7402) for 2 minutes,
followed by a brief wash with distilled water. After immersing the slides in
Bluing Reagent (Richard-Allan Scientific, 7301) for 1 minute, they were washed
with water (2 minutes), incubated in 95% ethanol for 1 minute, and then with
Eosin Y (Richard-Allan Scientific, 7111) for 20 seconds. Then, the slides were
incubated in 100% ethanol (three times, 1 minute each), followed by xylene
(three times, 1 minute each).
|
Apoptotic staining
Testes were fixed in 10% formalin (NBF; Sigma, HT50-1-128). Apoptotic
staining followed the manufacturer's protocol (Chemicon, S7100).
Immunoblotting and kinase assays
Whole-cell lysates from passage-three MEFs were prepared as described
(Berthet et al., 2003). For
western blotting, 50 µg of protein was separated on 12.5% polyacrylamide
gels (Bio-Rad), transferred onto Immobilon-P transfer membranes (Millipore,
IPVH00010) using semi-dry blotting, and probed with the following primary
antibodies: Cdk2, Cdk1, Cdk4, HA-Cdk2, cyclin B1 as described previously
(Berthet et al., 2003), cyclin
E1 (gift of Bruno Amati, European Institute of Oncology, Milan, Italy), cyclin
D1 (Neomarkers, RB-010p), p27 (Zymed, 71-9600) and actin (Santa Cruz, C0306).
All antibodies were used at 1:1000. For kinase assays (Cdk2 and HA-Cdk2), 250
µg of protein from cell lysates and 7 µl of anti-Cdk2 antibody-coupled
agarose A beads [as described by Berthet et al.
(Berthet et al., 2003)] or
HA-antibody-coupled agarose A beads (Roche, 11815016001) were used and the
kinase assays performed as described previously
(Aleem et al., 2005). For
co-immunoprecipitation assays (HA-Cdk2/cyclin E1, HA-Cdk2/cyclin A2), 400
µg of protein from cell lysates and 7 µl of HA-coupled agarose A beads
were used.
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). To
explore the reverse situation, i.e. whether Cdk2 can functionally substitute
for Cdk1, we knocked the Cdk2 cDNA into the Cdk1 locus
(Fig. 1A). The knockin
construct was electroporated into embryonic stem (ES) cells. The homologous
recombination event in heterozygous ES cells was identified by lacZ
reporter gene expression (Fig.
1B), PCR (Fig. 1C)
and Southern blot (data not shown). Heterozygous ES cells were injected into
mouse blastocysts to generate chimeras. The chimeric mice were backcrossed to
produce Cdk1+/Cdk2KI heterozygous mice. At this point,
mouse embryonic fibroblasts (MEFs) were prepared from E13.5 embryos of
Cdk1+/Cdk2KI mice and the expression and kinase activity
of knockin HA-tagged Cdk2 was analyzed. This analysis indicated that knockin
HA-Cdk2 was expressed and displayed kinase activity similar to that of
endogenous Cdk2 (Fig. 1D).
Heterozygous mice were then intercrossed to generate homozygous
Cdk1Cdk2KI/Cdk2KI mice. Out of 258 mice analyzed, 88 (34%)
were Cdk1+/+ and 170 (66%) were
Cdk1+/Cdk2KI, but no Cdk1Cdk2KI/Cdk2KI
mice were obtained. In addition, a total of 143 embryos were analyzed at E9.5
(n=23), E10.5 (n=19), E12.5 (n=23), E13.5
(n=35), E14.5 (n=16), E16.5 (n=13) and E18.5
(n=14). All embryos analyzed were either Cdk1+/+
or Cdk1+/Cdk2KI and none displayed the
Cdk1Cdk2KI/Cdk2KI genotype. Also, 69 blastocyts were
recovered from the uteri of four Cdk1+/Cdk2KI
super-ovulated females and from six Cdk1+/Cdk2KI naturally
bred females with Cdk1+/Cdk2KI male mice. None of these
blastocysts displayed any abnormal phenotypes, such as fragmentation,
shrinkage or degeneration, but all blastocysts genotyped were either
Cdk1+/+ or Cdk1+/Cdk2KI and none was
homozygous for Cdk1Cdk2KI. However, when we crossed
Cdk1+/Cdk2KI male or female mice with C57BL6 wild-type
mice, both wild-type and Cdk1+/Cdk2KI litters were
obtained at the expected frequency. This indicates that the
Cdk1Cdk2KI germ cells are viable and functional, but that
the fertilized homozygous embryos are unable to reach the blastocyst stage.
Our analysis suggests that the substitution of Cdk1 by Cdk2 leads to early
embryonic lethality before E3.5, comparable to the effect of deleting the
Cdk1 gene or a gene-trap mutation in the Cdk1 locus (our
unpublished results) (Santamaria et al.,
2007). To test whether the loss of p53 (Trp53 - Mouse Genome
Informatics) could rescue the phenotype of
Cdk1Cdk2KI/Cdk2KI mice, Cdk1+/Cdk2KI
mice were crossed with p53-/- mice. We did not obtain any
mice, embryos or blastocysts with the p53-/-
Cdk1Cdk2KI/Cdk2KI genotype (data not shown). This indicates
that loss of p53 does not rescue the phenotype of
Cdk1Cdk2KI/Cdk2KI mice and that at least one copy of
Cdk1 is essential for survival.
|
;
Ortega et al., 2003), even
though they express a functional copy of Cdk2 from the Cdk1
locus. The testes and ovaries of adult Cdk2-/-
Cdk1+/Cdk2KI mice were atrophic and were only about half the
size of those of Cdk2+/+ Cdk1+/Cdk2KI
mice (Fig. 2A,B). To determine
the cause of the sterility, a histological analysis was conducted on
post-natal day 90 (P90) ovaries of Cdk2-/-
Cdk1+/Cdk2KI and Cdk2+/+
Cdk1+/Cdk2KI mice. In Cdk2+/+
Cdk1+/Cdk2KI ovaries, the development of oocytes was
normal and the correct follicle stages were observed
(Fig. 2I,K). By contrast, in
Cdk2-/- Cdk1+/Cdk2KI ovaries, no follicles were
observed and the oocytes failed to develop in the atrophic ovaries, similar to
what is observed in Cdk2-/- females
(Fig. 2J,L)
(Berthet et al., 2003;
Ortega et al., 2003). A
histological analysis was also conducted on sexually immature and mature
testes of Cdk2-/- Cdk1+/Cdk2KI mice. This
revealed that in the testes of adult Cdk2-/-
Cdk1+/Cdk2KI mice (P90), the size of the seminiferous tubules
was much smaller than normal, resulting from a substantial depletion of
spermatocytes (Fig. 2C-F),
similar to that reported previously for Cdk2-/- mice
(Berthet et al., 2003;
Ortega et al., 2003). The
numbers of spermatogonia and Sertoli cells were not affected in testes of
Cdk2-/- Cdk1+/Cdk2KI mice
(Fig. 2F). In addition, we took
advantage of the lacZ reporter in the Cdk2 knockin construct
and analyzed the expression of knockin Cdk2 from the Cdk1 locus by
β-galactosidase staining in testes of Cdk2+/+
Cdk1+/Cdk2KI and Cdk2-/-
Cdk1+/Cdk2KI mice. We found that lacZ was abundantly
expressed in the testes of Cdk2+/+
Cdk1+/Cdk2KI mice and extensive β-galactosidase
staining was observed in the spermatocytes
(Fig. 2G). By contrast, in
Cdk2-/- Cdk1+/Cdk2KI mice, no expression was
detected in the seminiferous tubules owing to the substantial depletion of
spermatocytes (Fig. 2H), but
faint β-galactosidase staining was observed in the testes
(Fig. 2H).
In addition to adult testes, we also performed a histological analysis of
testes from P10 and P20 Cdk2+/+
Cdk1+/Cdk2KI and Cdk2-/-
Cdk1+/Cdk2KI mice. Hematoxylin and Eosin staining of P10
testes revealed no significant differences between Cdk2-/-
Cdk1+/Cdk2KI and Cdk2+/+
Cdk1+/Cdk2KI mice, which were similar to
Cdk2-/- and Cdk2+/+ mice,
respectively, as reported previously (Fig.
3Aa,b,Ba,b) (Ortega et al.,
2003). We observed a similar expression pattern of knockin Cdk2
(β-galactosidase staining) in the P10 testes of
Cdk2+/+ Cdk1+/Cdk2KI and
Cdk2-/- Cdk1+/Cdk2KI mice
(Fig. 3Ac,Bc). Nevertheless, we
detected a marked increase in apoptosis of primary spermatocytes in P10 testes
of Cdk2-/- Cdk1+/Cdk2KI as compared with
Cdk2+/+ Cdk1+/Cdk2KI mice
(Fig. 3Ad,Bd). In contrast to
P10 testes, visible defects were observed in P20 testes of
Cdk2-/- Cdk1+/Cdk2KI as compared with
Cdk2+/+ Cdk1+/Cdk2KI mice
(Fig. 3Ca,b,Da,b). At this
stage in development, the first wave of germ cells is completing the second
meiotic division and developing into round spermatids. Earlier stages of
spermatogenesis can also be detected in tubules of P20 mice. P20
Cdk2-/- Cdk1+/Cdk2KI testes were 20-30%
smaller than Cdk2+/+ Cdk1+/Cdk2KI
testes (data not shown). Histological analysis revealed the absence of round
spermatids in P20 Cdk2-/- Cdk1+/Cdk2KI testes,
similar to Cdk2-/- testes
(Fig. 3Da,b). In addition, we
found extensive germ cell apoptosis in P20 Cdk2-/-
Cdk1+/Cdk2KI as compared with Cdk2+/+
Cdk1+/Cdk2KI testes
(Fig. 3Cd,Dd). In accordance
with this germ cell apoptosis and depletion of spermatocytes, diminished
expression of knockin Cdk2 (β-galactosidase) was detected in P20
Cdk2-/- Cdk1+/Cdk2KI
(Fig. 3Dc) as compared with
Cdk2+/+ Cdk1+/Cdk2KI
(Fig. 3Cc) testes.
|
|
;
Satyanarayana et al., 2008).
By contrast, Cdk1 is present primarily in the cytoplasm and translocates to
the nucleus only during mitosis after nuclear breakdown
(Bailly et al., 1989;
Bailly et al., 1992). To
identify the consequences of genetic replacement for the translocational
properties of Cdk2 we monitored, by immunofluorescence, the localization of
endogenous Cdk2 and knockin Cdk2 (expressed from the Cdk1 locus) in
serum-starved and serum-stimulated Cdk2+/+
Cdk1+/Cdk2KI MEFs at different stages of the cell cycle
(Fig. 5Aa-Bh). Our analysis
revealed that endogenous Cdk2, as well as HA-tagged knockin Cdk2 expressed
from the Cdk1 locus, were predominantly present in the nucleus in
serum-starved cells, although we also found a certain amount of staining in
the cytoplasm for both endogenous and knockin Cdk2
(Fig. 5Ae,Be). Between 6 and 24
hours after serum stimulation, we detected the endogenous or knockin Cdk2
primarily localized in the nucleus (Fig.
5Af-Ah,Bf-Bh). Similarly, when we monitored the localization of
knockin Cdk2 in the absence of endogenous Cdk2 in Cdk2-/-
Cdk1+/Cdk2KI MEFs, we did not observe any difference in the
localization pattern of knockin Cdk2, as it was present predominantly in the
nucleus even though it was expressed from the Cdk1 locus (data not
shown).
To identify whether cells expressing three copies of Cdk2 had any
proliferative advantage over wild-type or Cdk2-/- MEFs, we
measured the proliferation rate of Cdk2+/+
Cdk1+/Cdk2KI, Cdk2+/+ and
Cdk2-/- MEFs. Our analysis indicated that there was no
significant difference in the proliferation rate of
Cdk2+/+ Cdk1+/Cdk2KI as compared with
Cdk2+/+ MEFs, even though they express an extra copy of
Cdk2 from the Cdk1 locus
(Fig. 5C). Similarly, we did
not observe any significant difference in the proliferation rate of
Cdk2-/- Cdk1+/Cdk2KI MEFs as compared with
Cdk2+/+ MEFs or those of the other two genotypes
(Fig. 5C).
Co-immunoprecipitation assays revealed that the knockin HA-tagged Cdk2 was
able to form a complex with cyclin E1 (Fig.
5D, eleventh panel from the top) and cyclin A2
(Fig. 5D, twelfth panel),
similar to endogenous Cdk2 as described previously
(Elledge et al., 1992;
Sheaff et al., 1997). In
addition, when we determined the expression pattern of some of the Cdks and
cyclins that play a role in the G1, S and G2 phases of the cell cycle, we did
not observe any significant differences in their expression levels between
Cdk2+/+, Cdk2-/-, Cdk2+/+
Cdk1+/Cdk2KI and Cdk2-/-
Cdk1+/Cdk2KI genotypes
(Fig. 5D), with the exception
of an increase in cyclin E expression when Cdk2KI was present
(Fig. 5D, lanes 3 and 4). This
indicates that the expression of Cdk2 from the Cdk1 locus did not
affect its cell cycle functions, and that the presence of an extra copy of
Cdk2, or the loss of one copy of Cdk1, does not have any
impact on the cell cycle.
|
;
Kountouras et al., 2001) to
study the transcriptional activation of Cdk1 during in vivo cell
cycle initiation and progression in the presence or absence of Cdk2
(Fig. 6Aa-Bd). In response to
PH, S-phase initiation occurs at 24 hours, peaks at 48 hours, and the first
round of replication is completed at 72 hours
(Fausto, 2000;
Satyanarayana et al., 2004).
In the presence of Cdk2 (Cdk2+/+
Cdk1+/Cdk2KI), initiation of Cdk1 transcription
occurred 24 hours after PH, as revealed by weak β-galactosidase staining
in the regenerating liver (Fig.
6Ab). At 48 hours after PH, β-galactosidase staining was
stronger than at 24 hours and 50% of the cells stained for
β-galactosidase (Fig.
6Ac). At 72 hours after PH, more than 90% of the cells displayed
β-galactosidase staining and the staining pattern was even stronger than
at earlier time points (Fig.
6Ad). This analysis suggests that the increase in the staining
pattern was due not only to increased transcriptional activation, but also to
the accumulation of more protein. In contrast to the transcriptional
activation of Cdk1 in Cdk2+/+
Cdk1+/Cdk2KI mice, Cdk2-/-
Cdk1+/Cdk2KI mice displayed premature transcriptional
activation of Cdk1 as revealed by a robust β-galactosidase
staining pattern at 24 hours after PH, when 40% of the cells already
stained for β-galactosidase (Fig.
6Bb). At later time points (48 hours after PH), the staining
pattern appeared more intense (70% of cells
β-galactosidase-positive) than in Cdk2+/+
Cdk1+/Cdk2KI mice (Fig.
6Bc). However, at 72 hours, the β-galactosidase staining
pattern was similar in Cdk2+/+
Cdk1+/Cdk2KI and Cdk2-/-
Cdk1+/Cdk2KI mice (Fig.
6Bd).
In addition to monitoring the transcriptional activation of Cdk1
by β-galactosidase staining, we also monitored the initiation and
progression of the cell cycle 24 to 72 hours after PH in
Cdk2+/+, Cdk2-/-, Cdk2+/+
Cdk1+/Cdk2KI and Cdk2-/-
Cdk1+/Cdk2KI mice. We observed that S-phase was slightly
delayed in Cdk2-/- as compared with
Cdk2+/+ mice, especially at 24 hours
(Fig. 6Cb,Db,E), as reported
previously (Satyanarayana et al.,
2008). The initiation and peak of S-phase were not altered, but
the percentage of BrdU-positive cells was decreased at 24 (and 48) hours after
PH in Cdk2-/- regenerating livers as compared with
Cdk2+/+ livers (Fig.
6Cb,E). In contrast to Cdk2-/- mice,
Cdk2-/- Cdk1+/Cdk2KI mice did not display any
difference in the regenerative response as compared with
Cdk2+/+ mice, and the percentage of BrdU-positive cells
between 24 and 72 hours after PH was similar to that of
Cdk2+/+ or Cdk2+/+
Cdk1+/Cdk2KI mice (Fig.
6Ca,c,d,Da,c,d,E). This indicates that the knockin Cdk2 expressed
from the Cdk1 locus is able to mimic the cell cycle function of
endogenous Cdk2. In addition, it appears that the presence of an extra copy of
Cdk2 in Cdk2+/+ Cdk1+/Cdk2KI
mice did not confer any proliferative advantage, and one copy of Cdk1
was sufficient for normal liver regeneration after PH. The differential
transcriptional activation of Cdk1 during different stages of liver
regeneration prompted us to explore the transcriptional activation of
Cdk1 during embryogenesis and in adult tissues.
|
Furthermore, when we analyzed the transcriptional activation of
Cdk1 by lacZ expression in several adult tissues in
Cdk2+/+ Cdk1+/Cdk2KI mice, we did not
observe expression of Cdk1 (β-galactosidase staining) in most of the
tissues, including brain, heart, liver, lung, kidney and skin
(Fig. 8A-E). In the case of the
thymus, β-galactosidase staining was mainly observed in the medulla
(Fig. 8F',F''). In
spleen, β-galactosidase staining was detected in the hematogenous red
pulp (Fig. 8G,G''). In
contrast to these other organs, robust expression of Cdk1 was observed in
testis: spermatids, spermatocytes and Sertoli cells were solidly stained for
β-galactosidase (Fig.
8H',H''). This observation is in accordance with
previous reports that Cdk1 is widely expressed in germ cells (see
Ravnik and Wolgemuth, 1999)
(see Fig. 4Aa,Ba,Ca,Da). When
we analyzed the expression level of endogenous Cdk2 and HA-tagged knockin Cdk2
in different tissues of adult Cdk2+/+
Cdk1+/Cdk2KI mice, expression was absent in most of the
adult tissues, except for spleen, testes and thymus
(Fig. 8I).
|
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Pagano and Jackson,
2004). Furthermore, it was suggested that the availability of
multiple Cdks in mammalian cells offers compensatory mechanisms in the absence
of one or more Cdks. In support of this hypothesis, a single deletion of
Cdk2, Cdk4 or Cdk6 does not affect the survival of mice
(Berthet et al., 2003;
Malumbres et al., 2004;
Ortega et al., 2003). In
addition, it was shown that double knockout of Cdk4 and
Cdk6, or of Cdk2 and Cdk4, leads to embryonic
lethality (Berthet et al.,
2006; Malumbres et al.,
2004; Santamaria et al.,
2007). These studies imply that the loss of one or two Cdks is
compensated partially or completely by other Cdk-cyclin complexes. Notably,
Cdk1 compensates for the loss of Cdk2 by complexing with cyclin E
(Aleem et al., 2005).
Nevertheless, compensation of Cdk2 by Cdk1 appears to be only partial as
Cdk2 knockout males and females are sterile
(Berthet et al., 2003;
Ortega et al., 2003). This
indicates that Cdk1 cannot fulfil the meiotic functions of Cdk2. To date,
whether any of the Cdks can substitute for the functions of Cdk1 has not been
explored. The fact that Cdk1, although a mitotic kinase, is able to perform
the functions of the S-phase kinase Cdk2, raises the possibility that Cdk2
might substitute for the loss of Cdk1. This hypothesis is strengthened further
by the observation that Cdk2 can bind to cyclin B1
(Aleem et al., 2005).
Contrary to this hypothesis, it has been reported that the deletion of
Cdk1 leads to early embryonic lethality, with embryos dying before
E3.5 (Santamaria et al., 2007)
(our unpublished results). This indicates that none of the Cdks can compensate
for the loss of Cdk1 in terms of lethality. We hypothesized that if the timing
of transcriptional activation and the genomic location of Cdk2 match
those of Cdk1, Cdk2 might acquire some of the properties of Cdk1 and
thereby compensate for the loss of Cdk1. However, even when Cdk2 was expressed
from the Cdk1 locus, we did not observe any rescue of the lethality.
We found that genetic replacement of Cdk1 by Cdk2 leads to early embryonic
lethality, similar to Cdk1 deletion, and embryos die before E3.5.
This indicates that Cdk1 is essential for the initial divisions that lead to
the formation of the blastocyst. In addition, deletion of p53 in the
knockin background did not rescue the phenotypes caused by the substitution of
Cdk1 by Cdk2. From this genetic replacement study, we were only able to obtain
heterozygous knockin mice (Cdk1+/Cdk2KI), in which one
copy of Cdk2 is expressed from the Cdk1 locus and the other
allele encodes wild-type Cdk1. Our work indicates that at least one
copy of Cdk1 is essential for the survival of mice and that Cdk2
cannot substitute for Cdk1 function, even when expressed from the
Cdk1 locus. Among the possible reasons for the failed rescue is that
the localization of Cdk2KI differs from that of Cdk1, although differences in
substrate specificity cannot be excluded either.
|
We observed that Cdk2-/- spermatocytes arrested and accumulated mostly prior to pachytene. This arrest appears to be incomplete, as we observed occasional cells with pachytene morphology. We believe that this arrest can be overcome by knockin Cdk2, as we see more cells with pachytene morphology in Cdk2-/- Cdk1+/Cdk2KI than in Cdk2-/- mice. It appears, however, that this rescue is only partial, as these spermatocytes arrest later in pachytene. Given that the subcellular localization of knockin Cdk2 appears to reflect that of the endogenous Cdk2, we conclude that the timing of expression of Cdk2 is crucial for its meiotic function(s). These results suggest the existence of a certain time window for the requirement of Cdk2. When Cdk2 is not expressed at that particular time point, the cells fail to complete meiosis even though Cdk2 is expressed subsequently, as indicated by the continuous HA and Cdk2 staining in the tubules of the Cdk2-/- Cdk1+/Cdk2KI mice after pachytene. Our results indicate that the genetic relocation of Cdk2 to the Cdk1 locus abolished Cdk2 meiotic function and as a result Cdk2-/- Cdk1+/Cdk2KI mice are sterile, similar to Cdk2-/- mice. This indicates that the genetic locus and timing of Cdk2 expression determine the meiotic functions of Cdk2.
When we analyzed the subcellular localization of knockin Cdk2, we found
that it was predominantly localized in the nucleus irrespective of the cell
cycle stage, similar to endogenous Cdk2
(Moore et al., 1999). Although
expressed from the Cdk1 locus, knockin Cdk2 retains its subcellular
localization. This indicates that the genomic locus does not play a
significant role in determining the translocational property of a protein, at
least in the case of Cdk2. Similarly, we found that knockin Cdk2 is able to
form a complex with cyclin E1 and cyclin A2 and displays kinase activity
similar to endogenous Cdk2. This excludes the possibility that the presence of
the HA tag affected the properties, and thereby meiotic function, of knockin
Cdk2. In addition, when we analyzed the proliferation rate of
Cdk2-/- Cdk1+/Cdk2KI MEFs, we did not observe
any significant difference to Cdk2+/+
Cdk1+/Cdk2KI MEFs. This indicates that the knockin Cdk2 is
able to perform its function in the mitotic cell cycle and form a complex with
cyclin E1. In addition, analysis of cell cycle initiation and progression in
vivo revealed that there was no significant difference between
Cdk2-/- Cdk1+/Cdk2KI and
Cdk2+/+ Cdk1+/Cdk2KI mice, indicating
that knockin Cdk2 was able to rescue the slight S-phase delay originally
identified in Cdk2-/- mice during liver regeneration.
Furthermore, we analyzed the transcriptional activation of the Cdk1
locus by lacZ reporter gene expression using liver regeneration as an
in vivo cell cycle model. This analysis revealed that Cdk1
transcriptional activation occurred earlier in the absence of Cdk2, suggesting
that premature activation of Cdk1 is essential in the absence of Cdk2
in order to promote the G1-S transition. This observation is in accordance
with our recent finding that Cdk1, as judged by protein level, is induced at
an earlier time point in the absence of Cdk2
[(Satyanarayana et al., 2008),
see Fig. 5C therein]. It
appears that premature transcriptional and translational activation of
Cdk1 are essential in the absence of Cdk2 to drive cells through the
G1-S transition by binding to cyclin E. In this context, it will be
interesting to determine which molecular mechanisms are responsible for
coordinating the transcriptional activation of Cdk1 and
Cdk2. When we analyzed the transcriptional activation of
Cdk1 in adult tissues by lacZ expression, we did not observe
β-galactosidase staining in most of the tissues. This might be due to the
fact that most of the adult organs are quiescent and mitotically inactive. By
contrast, we found solid transcriptional activation of Cdk1 during
different stages (E14.5 to E20.5) of embryogenesis. Our results suggest that
Cdk1 is essential for the differentiation and development of various organs
during embryogenesis.
The present study indicates that Cdk1 is essential for the survival of mice. Genetic substitution of Cdk1 by Cdk2 leads to early embryonic lethality. This indicates that Cdk2 cannot substitute for the loss of Cdk1, even when the timing of transcription and genetic location of Cdk2 match those of Cdk1. Most interestingly, Cdk2 loses its meiotic function when expressed from the Cdk1 locus, even though it is able to perform its mitotic cell cycle functions by complexing with cyclin E1 and cyclin A2. In addition, an increase in the transcriptional activation of Cdk1 during late embryogenesis (E14.5 to E20.5) indicated that Cdk1 is not only essential for early embryogenesis, but might also be essential in the latter stages of embryogenesis.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/20/3389/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Aleem, E. and Kaldis, P. (2006). Mouse models of cell cycle regulators: new paradigms. Results Probl. Cell Differ. 42,271 -328.[Medline]
Aleem, E., Kiyokawa, H. and Kaldis, P. (2005). Cdc2-cyclin E complexes regulate the G1/S phase transition. Nat. Cell Biol. 7,831 -836.[CrossRef][Medline]
Bailly, E., Dorée, M., Nurse, P. and Bornens, M. (1989). p34cdc2 is located in both nucleus and cytoplasm; part is centrosomally associated at G2/M and enters vesicles at anaphase. EMBO J. 8,3985 -3995.[Medline]
Bailly, E., Pines, J., Hunter, T. and Bornens, M.
(1992). Cytoplasmic accumulation of cyclin B1 in human cells:
association with a detergent-resistant compartment and with the centrosome.
J. Cell Sci. 101,529
-545.
Berthet, C., Aleem, E., Coppola, V., Tessarollo, L. and Kaldis, P. (2003). Cdk2 knockout mice are viable. Curr. Biol. 13,1775 -1785.[CrossRef][Medline]
Berthet, C., Klarmann, K. D., Hilton, M. B., Suh, H. C., Keller, J. R., Kiyokawa, H. and Kaldis, P. (2006). Combined loss of Cdk2 and Cdk4 results in embryonic lethality and Rb hypophosphorylation. Dev. Cell 10,563 -573.[CrossRef][Medline]
Carthon, B. C., Neumann, C. A., Das, M., Pawlyk, B., Li, T.,
Geng, Y. and Sicinski, P. (2005). Genetic replacement of
cyclin D1 function in mouse development by cyclin D2. Mol. Cell.
Biol. 25,1081
-1088.
Dunphy, W. G., Brizuela, L., Beach, D. and Newport, J. (1988). The Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis. Cell 54,423 -431.[CrossRef][Medline]
Elledge, S. J., Richman, R., Hall, F. L., Williams, R. T.,
Lodgson, N. and Harper, J. W. (1992). CDK2 encodes a
33-kDa cyclin A-associated protein kinase and is expressed before
CDC2 in the cell cycle. Proc. Natl. Acad. Sci.
USA 89,2907
-2911.
Fausto, N. (2000). Liver regeneration. J. Hepatol. 32,19 -31.[Medline]
Geng, Y., Whoriskey, W., Park, M. Y., Bronson, R. T., Medema, R. H., Li, T., Weinberg, R. A. and Sicinski, P. (1999). Rescue of cyclin D1 deficiency by knockin cyclin E. Cell 97,767 -777.[CrossRef][Medline]
Izumi, T. and Maller, J. L. (1993). Elimination of cdc2 phosphorylation sites in the cdc25 phosphatase blocks initiation of M-phase. Mol. Biol. Cell 4,1337 -1350.[Abstract]
Kountouras, J., Boura, P. and Lygidakis, N. J. (2001). Liver regeneration after hepatectomy. Hepatogastroenterology 48,556 -562.[Medline]
Lee, E.-C., Yu, D., Martinez de Velasco, J., Tessarollo, L., Swing, D. A., Court, D. L., Jenkins, N. A. and Copeland, N. G. (2001). A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics 73, 56-65.[CrossRef][Medline]
Liu, P., Jenkins, N. A. and Copeland, N. G.
(2003). A highly efficient recombineering-based method for
generating conditional knockout mutations. Genome Res.
13,476
-484.
Malumbres, M., Sotillo, R., Santamaria, D., Galan, J., Cerezo, A., Ortega, S., Dubus, P. and Barbacid, M. (2004). Mammalian cells cycle without the D-type cyclin-dependent kinases Cdk4 and Cdk6. Cell 118,493 -504.[CrossRef][Medline]
Mittnacht, S. (1998). Control of pRB phosphorylation. Curr. Opin. Genet. Dev. 8, 21-27.[CrossRef][Medline]
Moore, J. D., Yang, J., Truant, R. and Kornbluth, S.
(1999). Nuclear import of Cdk/Cyclin complexes: identification of
distinct mechanisms for import of Cdk2/Cyclin E and Cdc2/Cyclin B1.
J. Cell Biol. 144,213
-224.
Morgan, D. O. (1997). Cyclin-dependent kinases: engines, clocks, and microprocessors. Annu. Rev. Cell Dev. Biol. 13,261 -291.[CrossRef][Medline]
Nasmyth, K. (1993). Control of the yeast cell cycle by the Cdc28 protein kinase. Curr. Opin. Cell Biol. 5,166 -179.[CrossRef][Medline]
Ortega, S., Prieto, I., Odajima, J., Martin, A., Dubus, P., Sotillo, R., Barbero, J. L., Malumbres, M. and Barbacid, M. (2003). Cyclin-dependent kinase 2 is essential for meiosis but not for mitotic cell division in mice. Nat. Genet. 35, 25-31.[CrossRef][Medline]
Pagano, M. and Jackson, P. K. (2004). Wagging the dogma; tissue-specific cell cycle control in the mouse embryo. Cell 118,535 -538.[CrossRef][Medline]
Pan, Z. Q., Amin, A. and Hurwitz, J. (1993).
Characterization of the in vitro reconstituted cyclin A or B1-dependent Cdk2
and Cdc2 kinase activities. J. Biol. Chem.
268,20443
-20451.
Pines, J. and Hunter, T. (1991). Human cyclins
A and B1 are differentially located in the cell and undergo cell
cycle-dependent nuclear transport. J. Cell Biol.
115, 1-17.
Potenza, N., Vecchione, C., Notte, A., De Rienzo, A., Rosica, A., Bauer, L., Affuso, A., De Felice, M., Russo, T., Poulet, R. et al. (2005). Replacement of K-Ras with H-Ras supports normal embryonic development despite inducing cardiovascular pathology in adult mice. EMBO Rep. 6,432 -437.[CrossRef][Medline]
Ravnik, S. E. and Wolgemuth, D. J. (1999). Regulation of meiosis during mammalian spermatogenesis: the A-type cyclins and their associated cyclin-dependent kinases are differentially expressed in the germ-cell lineage. Dev. Biol. 207,408 -418.[CrossRef][Medline]
Reed, S. I., Ferguson, J. and Groppe, J. C.
(1982). Preliminary characterization of the transcriptional and
translational products of the Saccharomyces cerevisiae cell division
cycle gene CDC28. Mol. Cell. Biol.
2, 412-425.
Riabowol, K., Draetta, G., Brizuela, L., Vandre, D. and Beach, D. (1989). The cdc2 kinase is a nuclear protein that is essential for mitosis in mammalian cells. Cell 57,393 -401.[CrossRef][Medline]
Santamaria, D., Barriere, C., Cerqueira, A., Hunt, S., Tardy, C., Newton, K., Caceres, J. F., Dubus, P., Malumbres, M. and Barbacid, M. (2007). Cdk1 is sufficient to drive the mammalian cell cycle. Nature 448,811 -815.[CrossRef][Medline]
Satyanarayana, A., Wiemann, S. U., Buer, J., Lauber, J., Dittmar, K. E., Wustefeld, T., Blasco, M. A., Manns, M. P. and Rudolph, K. L. (2003). Telomere shortening impairs organ regeneration by inhibiting cell cycle re-entry of a subpopulation of cells. EMBO J. 22,4003 -4013.[CrossRef][Medline]
Satyanarayana, A., Geffers, R., Manns, M. P., Buer, J. and Rudolph, K. L. (2004). Gene expression profile at the G1/S transition of liver regeneration after partial hepatectomy in mice. Cell Cycle 3,1405 -1417.[Medline]
Satyanarayana, A., Hilton, M. B. and Kaldis, P.
(2008). p21 inhibits Cdk1 in the absence of Cdk2 to maintain the
G1/S phase DNA damage checkpoint. Mol. Biol. Cell
19, 65-77.
Sheaff, R. J., Groudine, M., Gordon, M., Roberts, J. M. and
Clurman, B. E. (1997). Cyclin E-CDK2 is a regulator of
p27Kip1. Genes Dev.
11,1464
-1478.
Weinberg, R. A. (1995). The Retinoblastoma protein and cell cycle control. Cell 81,323 -330.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
Related articles in Development:
This article has been cited by other articles:
|
|
 |
|
  A. Viera, J. S. Rufas, I. Martinez, J. L. Barbero, S. Ortega, and J. A. Suja CDK2 is required for proper homologous pairing, recombination and sex-body formation during male mouse meiosis J. Cell Sci., June 15, 2009; 122(12): 2149 - 2159. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Li, S. Kotoshiba, C. Berthet, M. B. Hilton, and P. Kaldis Rb/Cdk2/Cdk4 triple mutant mice elicit an alternative mechanism for regulation of the G1/S transition PNAS, January 13, 2009; 106(2): 486 - 491. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
