3083
Journal of Cell Science 110, 3083-3090 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
JCS1511
A casein kinase I isoform is required for proper cell cycle progression in the
fertilized mouse oocyte
Stefan D. Gross1,2, Calvin Simerly3, Gerald Schatten2,3 and Richard A. Anderson1,2,*
1Department of Pharmacology, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI 53706, USA
2Cellular and Molecular Biology Program, University of Wisconsin, Madison, WI 53706, USA
3Departments of Zoology and Obstetrics-Gynecology, University of Wisconsin, 1117 W. Johnson St, Madison, WI 53706, USA
*Author for correspondence (e-mail: [email protected])
SUMMARY
Casein kinase I is a family of serine/threonine protein kinases
common to all eukaryotes. In yeast, casein kinase I homologues have been linked to the regulation of growth, DNA
repair and cell division. In addition, their subcellular localization to membraneous structures and the nucleus is
essential for function. In higher eukaryotes, there exist seven
genetically distinct isoforms: α, β, γ1, γ2, γ3, δ and ε. Casein
kinase Iα exhibits a cell cycle-dependent subcellular localization including an association with cytosolic vesicular
structures and the nucleus during interphase, and the
spindle during mitosis. casein kinase I has also been shown
to modulate critical regulators of growth and DNA
synthesis/repair in mammalian cells such as SV40 large T
antigen and p53. These results suggest that casein kinase I
may be involved in processes similar to those ascribed to the
yeast casein kinase I homologues. To define a role for casein
kinase Iα in cell cycle regulation, the mouse oocyte was
utilized because of its well-defined cell cycle and ease of
micromanipulation. Immunofluorescence studies from
meiosis I of maturation to the first zygotic cleavage demonstrated that the kinase was associated with structures similar
to those previously reported. Microinjection of casein kinase
Iα antibodies at metaphase II-arrest and G2 phase, had no
effect on the completion of second meiosis or first division.
However, microinjection of these antibodies during the early
pronucleate phase prior to S-phase onset blocked uptake of
the kinase into pronuclei and interfered with proper and
timely cell cycle progression to first cleavage. These results
suggest that the kinase regulates the progression from interphase to mitosis during the first cell cycle.
INTRODUCTION
otes there exist at least seven genetically distinct isoforms of
CKI termed α, β, γ1, γ2, γ3, δ and ε (Fish et al., 1995; Graves
et al., 1993; Rowles et al., 1991; Zhai et al., 1995). As a family,
these isoforms occupy their own unique branch on the kinase
phylogenetic tree because they are greater than 50% identical
to one another and less than 25% identical to any other known
kinase (Fish et al., 1995; Graves et al., 1993; Rowles et al.,
1991; Zhai et al., 1995). These molecular genetic data subsequently led to the identification of four CKI homologues each
in both S. cerevisiae and S. pombe (DeMaggio et al., 1992;
Dhillon and Hoekstra, 1994; Hoekstra et al., 1991; Kearney et
al., 1994; Robinson et al., 1992; Wang et al., 1992). The
budding yeast isoforms HRR25, YCK1, YCK2 and YCK3
(Hoekstra et al., 1991; Robinson et al., 1992; Wang et al., 1996)
are the most characterized.
HRR25 was isolated in a screen for mutants with increased
sensitivity to the constuitive expression of HO endonuclease,
or treatment with alkylating agents or x-irradiation all of which
induce DNA strand breakage (Hoekstra et al., 1991). The associated hrr25 phenotype involved defects in a number of
processes including meiosis, mitosis, chromosomal segregation as well as a cell cycle delay in G2 (Hoekstra et al., 1991).
These defects, it was supposed, were due to aberrations in some
checkpoint which interfered with the ability of the mutant to
The growth cycle of eukaryotic cells is divided into four
distinct phases termed G1, S, G2 and M. Occurring within each
of these phases are specific metabolic processes allowing the
cell to attain the appropriate size, replicate its DNA and
segregate its genetic material equally among its daughter cells.
Naturally, all of these processes are stringently regulated to
ensure proper completion before further progression through
the cell cycle is allowed. One of the primary players in cell
cycle progression regulation is the family of cyclin-dependent
kinases or cdks of which eight higher eukaryotic isoforms have
been identified so far (Grana and Reddy, 1995; Machlachlan et
al., 1995; Nigg, 1996). In yeast, these serine/threonine kinases
play direct roles in the regulation of progression through all of
the cell cycle phases (Grana and Reddy, 1995; Pines, 1994).
However, as the complexities of cell cycle regulation unravel,
it is clear that other kinase activities are providing essential
functions in these regulatory cascades providing an additional,
finer level of control.
One such kinase activity is the casein kinase I (CKI) family
of serine/threonine protein kinases common to all eukaryotic
systems from yeast to humans (Tuazon and Traugh, 1991).
Recent molecular genetic data has shown that in higher eukary-
Key words: Casein kinase I, Mouse oocyte, Protein kinase, Cell cycle
3084 S. D. Gross and others
respond to DNA strand breakage. Sequence analysis of HRR25
established that it was 64% identical to CKIα through the
catalytic domain (DeMaggio et al., 1992). In addition,
mutations in Hrr25p’s active site lysine, a residue essential for
the activity of a number of kinases, resulted in the same
phenotype as the null mutant (Hoekstra et al., 1991). A recent
report has demonstrated that Hrr25p associates with and phosphorylates SBF, a transcription factor not only involved in the
transcriptional response to DNA damage, but also in the transcription of some G1 cyclins (Ho et al., 1997). This evidence
suggests that HRR25 may be involved in normal cell cycle progression in addition to the DNA damage response. YCK3, in
conjunction with HRR25, comprise an essential gene pair
(Wang et al., 1996). The two isoforms are predominantly
nuclear and their localization to this compartment is absolutely
required for function (Wang et al., 1996).
As for YCK1p and 2, they have been shown to comprise the
vast majority of casein kinase activity in S. cerevisiae (Robinson
et al., 1993). Knocking out either gene generates no readily
apparent phenotype (Robinson et al., 1993). However, interruption of both genes is lethal (Robinson et al., 1992). To date, no
direct function has been ascribed to YCK1. On the other hand,
YCK2 has been genetically linked to processes involved in bud
site selection, bud morphogenesis and cytokinesis (Robinson et
al., 1993). In addition, a recent report has linked these CKI
isoforms to the regulation of growth resumption after nutrient
re-addition as well as some aspect(s) of endo/exocytosis (Wang
et al., 1996). Sequence analysis of their respective genes determined that these isoforms are 56% identical to CKIα through
the catalytic domain (Graves et al., 1993; Robinson et al., 1993).
Furthermore, these isoforms contain an isoprenylation site at
their C terminus that is essential for their function (Robinson et
al., 1993; Vancura et al., 1994; Wang et al., 1996). Cell fractionation studies indicate that these enzymes partition exclusively in the membraneous fraction further supporting the contention that they are covalently attached to the inner leaflet of
the plasma membrane (Vancura et al., 1994 ; Wang et al., 1996).
Two significant observations can be made from these genetic
studies in yeast regarding the function(s) of CKI in cells. First,
these isoforms are likely involved in the regulation cascades of
several cell cycle checkpoints including G0/G1, S/DNA damage,
and mitosis. Secondly, the subcellular localization of these
isoforms is critical to their functioning. Because of the high
degree of conservation between the yeast and higher eukaryotic
isoforms, it is likely that these same conclusions will hold for
all members of the CKI family of kinases. However, the mechanisms by which the higher eukaryotic isoforms are segregated
into specific cellular compartments and the functions that they
provide will likely prove more diverse and complex.
As for the higher eukaryotic isoforms, CKIα has been shown
to possess a cell cycle-dependent subcellular localization.
During interphase, CKIα is associated with cytosolic vesicular
structures and the centrosome (Brockman et al., 1992). During
mitosis, CKIα is associated with the mitotic spindle (Brockman
et al., 1992). In addition, CKIα appears to associate with a
distinct subset of cytosolic vesicles, including small synaptic
vesicles, and can phosphorylate a specific subset of synaptic
vesicle-associated proteins (Gross et al., 1995). These observations suggest that, in higher eukaryotes, CKIα may be performing roles similar to those ascribed to the yeast isoforms. Association with the mitotic spindle suggests a role in
mitosis/cytokinesis while a vesicular localization implies a role
in vesicular trafficking. In yeast, disruption of both these
functions causes cells to exhibit cell cycle-related defects as well
(Hoekstra et al., 1991; Robinson et al. 1993; Wang et al., 1996).
In the studies described here, a mouse oocyte developmental system was employed to link CKI both temporally and
spatially to a particular function within the context of the cell
cycle. This experimental system possesses a number of advantages over established cell lines. First, mouse oocytes are
exceptionally large allowing for relative ease of micromanipulation. Second, at ovulation they are naturally arrested at
metaphase of meiosis II thereby allowing for its controlled progression through subsequent phases of the cell cycle by fertilization or parthenogenetic activation. Finally, the mouse oocyte
cell cycle is relatively slow, permitting the precise temporal
mapping of CKI’s potential involvement in any number of cell
cycle-related events. Using the mouse oocyte, we have established that CKIα is associated with cytoplasmic cytasters,
microtubule organizing centers structurally and functionally
related to centrosomes, and spindles, thereby reconfirming
previous observations in established cell lines. We also
observed that CKI was exclusively associated with distinct
structures within the pronuclei of the one-cell stage embryo.
Microinjection of CKI-specific antibodies into living oocytes
at various developmental stages from the onset of mitosis of
meiosis I through the first zygotic cleavage, established a
requirement for CKI in the early pronucleate phase 0-8 hours
after fertilization to ensure proper cell cycle progression.
MATERIALS AND METHODS
Materials
Rabbit polyclonal antibodies to CKIα were synthesized and characterized as previously described (Brockman et al., 1992; Gross et al.,
1995; Zhang et al., 1996). Mouse monoclonal antibodies to β-tubulin
were obtained from the Iowa Hybridoma Bank (E7; Iowa). Fluorescein and rhodamine-conjugated goat anti-rabbit secondary antibodies were purchased from Zymed Antibodies, CA.
CKI-immunodepleted IgGs were prepared by repeatedly cycling
the rabbit immune sera over a CKIα affinity column until no
detectable levels of IgG were eluted by the application of 0.1 M
glycine, pH 2.5 (5-8 cycles). This depleted sera was then applied to a
Protein A-Sepharose column to purify the remaining IgGs by subsequent elution with 0.1 M glycine, pH 2.5.
Western blotting
Cell lysates were prepared by the addition of 4 times concentrated
SDS-containing Laemmli sample buffer and subsequent boiling. A
volume equaling 300 mouse oocytes and approximately 1×105 Balb
3T3 cells were then loaded in their respective lanes and run out on a
12% SDS-polyacrylamide gel using a Bio-Rad protean II minigel
apparatus. The separated proteins were transferred to nitrocellulose
and air-dried. Any non-specific binding sites were blocked with a
solution containing PBS and 5% (w/v) non-fat dry milk for 18 hours
at 4°C. The blot was incubated with CKIα affinity-purified antibodies
in a PBS solution containing 3% BSA (w/v) for 2 hours at room temperature. The membrane was subsequently washed three times in PBS
with 0.5% Tween-20 and incubated for 1 hour with 125I-Staph A
protein (5 µCi/ml) in PBS with 3% BSA (w/v) at 23°C. The
membrane was washed overnight in PBS containing Tween-20 as
above. After drying, the blot was exposed to film.
Immunoprecipitation
Approximately 300 mouse oocytes were lysed for 1 hour at 4°C in a
CKI in the mouse oocyte cell cycle 3085
buffer containing 10 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.5%
Triton X-100, 2 mM PMSF, 100 µM leupeptin, and 10 µg/ml aprotinin
(Stefanovic and Maller, 1988; Gavin and Schorderetslatkine, 1997).
The lysates were then supplemented with a PBS solution containing
3% BSA to a final concentration of 1% BSA. 100 µg of Dynabeadstm
conjugated with goat anti-rabbit IgGs were added to each sample and
incubated for 1 hour at 4°C with constant mixing by inversion. The
beads were then removed based upon their ability to be isolated with
the application of a magnetic field. One of the two lysates was then
supplemented with 500 ng CKIα antibody and incubated along with
the other lysate for 2 hours at 4°C with mixing. Both lysates received
200 µg Dynabeadstm and were incubated for 2 hours with mixing. The
beads were then isolated as above and washed 3 times with phosphorylation buffer (50 mM Tris, pH 7.5, 2 mM MgCl2) and used as a
source of kinase in the activity assays described below.
Kinase activity assays
The Dynabeads containing kinase or the control were resuspended in
50 µl of phosphorylation buffer containing 40 µg Phosvitin. The two
reactions were carried out at room temperature for 30 minutes at room
temperature with mixing every 5 minutes. The reactions were quenched
with the addition of 4 times concentrated Laemmli SDS sample buffer
and the resultant samples were then boiled for 10 minutes. The samples
were loaded on a 12% SDS-polyacrylamide gel and separated as
described in the western blotting section. The gel was then stained with
Coomassie and destained overnight to reduce background noise. The
gel was dried down using a Hoefer gel drying apparatus and exposed
to film. The bands corresponding to substrate were excised and the
amount of radioactivity quantified by scintillation counting.
Animal handling and gamete collection
Superovulation, collection of mature unfertilized oocytes, mating of
the stimulated mouse ICR females and collection of immature GVstage oocytes from ovaries were accomplished as previously described
(Simerly et al., 1990). Cumulus cells were removed by treatment with
hyaluronidase. The zona was eliminated with a brief incubation in
acid Tyrodes, pH 2.5 (Simerly and Schatten, 1993).
Immunofluorescence microscopy
Immunfluorescence procedures were performed essentially as described
elsewhere (Simerly and Schatten, 1993). In brief, for detection of CKIα
and microtubules (MTs), mouse oocytes and zygotes were prepared for
indirect immunofluorescence by permeablization in a microtubule stabilization buffer containing 50 mM imidazole, pH 6.8, 50 mM KCl, 0.5
mM MgCl2-6H2O, 1 mM EGTA, 0.1 mM EDTA, 1 mM β-mercaptoethanol, 25% glycerol, followed by methanol fixation. Microtubules
were labeled with a mouse monoclonal antibody against β-tubulin and
casein kinase was detected with a rabbit polyclonal antibody which was
raised against the 37.5 kDa CKIα isoform purified from human erythrocyte cytosol. DNA was detected with DAPI in the penultimate PBS
rinse. Appropriate secondary antibodies conjugated to fluorescein or
rhodamine were used to detect both MTs and CKIα in the same oocytes
or zygote with double-labeling protocols. Conventional epifluorescence
microscopy was accomplished using a Zeiss Axiophot equipped with
appropriate chromophore filters.
Microinjection of CKIα antibody and immunofluorescence
detection
Micropipettes were front-loaded with 7.5 mg/ml affinity-purified
CKIα specific antibodies in microinjection buffer (20 mM NaCl, 115
mM KCl, 10 mM Hepes, 1 mM MgCl2, pH 7.0) from a droplet under
mineral oil juxtaposed to the culture medium containing the oocytes.
Microinjection was performed by puncturing zona-intact oocytes with
a 1-2 µm bevelled micropipette, sucking in a small amount of
cytoplasm, and expelling the antibody and cytoplasm back into the
cell. Typically, ~5% of the egg volume was microinjected into oocytes
and zygotes. To localize CKIα in microinjected cells, oocytes or
zygotes were labeled with goat anti-rabbit secondary antibody without
further primary antibody application. Application of secondary antibodies alone or after microinjection 7.5 mg/ml CKIα Ab-depleted
rabbit IgG did not result in MT or CKIα detection.
Parthenogenetic activation of unfertilized oocytes
Meiotic resumption of mature metaphase II-arrested oocytes was
accomplished by activation with 7% ethanol in culture medium for 7
minutes (Kaufman, 1983).
RESULTS
CKIα is present and active within mouse oocytes
Mouse oocytes arrested at metaphase II were western blotted
with antibodies specific for CKIα to determine whether the
kinase was being expressed in this system. The specificity of this
antibody for CKIα has been extensively defined for a number of
different species, including mouse (Brockman et al., 1992; Gross
et al., 1995; Zhang et al., 1996). As shown in Fig. 1, a single
immunoreactive band of 37 kDa is evident both in whole cell
lysates derived from mouse Balb 3T3 cells and mouse oocytes.
This apparent molecular mass is of the size expected for CKIα
(Rowles et al., 1991). CKI was then immunoprecipitated from
lysates derived from either metaphase II or fertilized oocytes and
analyzed for activity towards phosvitin, a good CKI substrate.
Results indicated that the CKIα antibodies immunoprecipitated
similar amounts of phosvitin kinase activity from both lysates
(Fig. 1B). Fig. 1B depicts measurements of 32P incorporation
into phosvitin as described in Materials and Methods.
Indirect immunofluorescence analysis of the
subcellular localization of CKIα throughout early
mouse development
The subcellular distribution of CKIα within the mouse oocyte
from meiosis I of meiotic maturation through metaphase of
meiosis II up to the completion of the first zygotic cleavage
was then examined to define the development-dependent subcellular distribution of CKIα.
Indirect immunofluorescence using antibody probes specific
for CKIα on germinal vesicle stage oocytes (Fig. 2A) indicated
that the kinase was distributed heterogeneously. CKIα (Fig.
2C) was not detected in the germinal vesicle (Fig. 2B, inset)
nor any cytosolic structures including the cytasters as defined
by β-tubulin staining (Fig. 2B, arrows). The kinase was,
however, clearly associated with the plasma membrane. During
meiotic maturation, the kinase was undetectable on the first
meiotic spindle apparatus (not shown), however, when the
oocytes arrest at second meiotic metaphase (Fig. 2D), CKIα
was clearly associated with the spindle (Fig. 2F) as defined by
tubulin staining (Fig. 2E). There was no obvious association of
the kinase with the plasma membrane except at the microvilli
deficient area (MVA, Fig. 2F; arrow) located immediately
above the spindle apparatus. When CKIα antibodies were
microinjected into metaphase II-arrested oocytes, fixed and
prepared for immunofluorescence, the kinase was also
observed associated with cytoplasmic cytasters (Fig. 3C,
arrow), structurally and functionally similar to centrosomes, in
addition to the spindle.
Eight hours after fertilization, CKIα had relocalized from the
spindle apparatus, MVA and cytasters to the newly formed male
and female pronuclei (Fig. 2G-I). CKIα (Fig. 2I) was associated
3086 S. D. Gross and others
B
with discrete pronuclear structures that also contained DNA
(Fig. 2H, inset) but was excluded from the nucleoli. The kinase
was essentially undetectable on any other structure within the
oocyte including the plasma membrane. As the oocytes progressed through their DNA synthesis and G2 phase, CKI
remained associated with these pronucleate structures. During
first mitosis (Fig. 2J), CKI was again readily apparent on the
spindle (Fig. 2L) observed with tubulin antibodies (Fig. 2K).
Interestingly, after first cleavage (Fig. 2M), CKI did not immediately relocalize to the daughter nuclei of cleaved zygotes (Fig.
2O) but instead remained distributed throughout the cytosol with
an observed accumulation at or around the nuclear envelope.
Microinjection of CKIα antibodies into mouse
oocytes interferes with cell cycle progression
To define a role for CKIα in the regulation of early mouse activation and development, CKIα antibodies were microinjected
into mouse oocytes or zygotes. The effects of these antibodies
on spindle assembly, chromosomal congression and segregation during meiosis and at first zygotic cleavage were then
observed. Microinjection of CKIα antibodies into GV-arrested
ooctyes did not affect spindle formation, chromosomal congression or segregation at first meiosis (not shown). When
CKIα antibodies were microinjected into metaphase IIarrested oocytes and observed at 5 hours post-activation,
defects in spindle re-orientation, chromosomal sorting, second
polar body formation or the appearance of female pronuclei
were not apparent (Fig. 3A-F). In addition, when G2 phase (2628 hours post-hCG) one-cell embryos were microinjected with
CKIα or control IgGs and followed out to first cleavage, no
defects were observed in spindle formation, chromosome condensation and congression or cytokinesis (not shown).
When fertilized mouse oocytes were microinjected with
CKIα antibodies at 20 hours post hCG, at or near the time of
sperm incorporation, a defect in subsequent development was
readily apparent. These results are depicted in Fig. 4. At 39
hours post hCG, a time at which more than 80% of the control
fertilized oocytes had undergone first cleavage, only 37% of
oocytes injected with CKIα antibodies had progressed to the
6000
5000
Fertilized
Met II
4000
3000
2000
1000
32
Fig. 1. Western blot using antibodies to CKIα and a
kinase activity assay with CKI immunoprecipitated from
mouse oocytes. (A) Western blot of Balb 3T3 cells and
metaphase II-arrested mouse oocytes demonstrating that
a 37 kDa immunoreactive band is detected in both
samples. (B) Kinase activity assay using phosvitin as
substrate and CKI immunoprecipitated from metaphase
II-arrested and fertilized oocytes, collected at 24 hours
post-hCG corresponding to S or early G2 phase (see
Materials and Methods).
P incorporated into phosvitin (cpm)
Immunoprecipitated CKI activity
0
two-cell stage. Those developmentally blocked zygotes did not
enter mitosis but rather locked up at the G2/M transition. As
an additional negative control, CKIα-immunodepleted IgGs
(see Materials and Methods) were injected at the identical concentration. These probes also failed to produce any noticeable
aberration in zygotic development in that, like the mockinjected oocytes, more than 80% of the immuno-depleted IgGinjected oocytes had undergone first cleavage at 39 hours posthCG. When the treated oocytes were analyzed 43 hours
post-hCG, >90% of mock-injected and more than 80% of the
immuno-depleted controls had undergone first cleavage and
were now in the 2-cell stage. On the other hand, only 40% of
the oocytes receiving the CKIα antibodies had progressed to
the two-cell stage. Those CKIα-antibody-injected oocytes that
did not arrest completed pronuclear formation, apposition and
centration correctly and entered into mitosis and completed
cleavage normally.
Microinjection of CKIα antibodies prevents the
importation of the kinase into the pronuclei
One possible means by which the introduction of CKIα antibodies into early mouse oocytes could be eliciting their effect
is by interfering with the kinase’s ability to relocalize to the
male and female pronuclei. One could then envision a scenario
in which the kinase is prevented from interacting and therefore
modulating the activities of substrates critical to proper and
timely cell cycle progression. To ascertain whether such a
blockade was occurring, mouse oocytes arrested at metaphase
II were microinjected with control IgGs or CKIα antibodies and
then parthenogenetically activated as described in Materials and
Methods. At 9 hours post-activation, the cells were prepared for
immunofluorescence. The oocytes were incubated with primary
antibodies to CKIα in addition to fluorescently conjugated
secondary antibodies. In those oocytes receiving immunodepleted antibodies (Fig. 5A), CKI was readily apparent in the
female pronucleus (Fig. 5B) in a distribution similar to that
described previously (Fig. 2I). However, those oocytes microinjected with CKIα antibodies (Fig. 5C) completely lacked any
signal for the kinase in the pronuclei (Fig. 5D).
CKI in the mouse oocyte cell cycle 3087
Fig. 2. Localization of CKIα in
the developing mouse oocyte
from the germinal vesicle stage
through the first zygotic
cleavage. Oocytes were fixed at
various developmental stages
(A,D,G,J,M, differential
interference contrast (DIC)) and
prepared for
immunofluorescence using
tubulin antibodies (B,E,H,K,N),
CKIα-specific antibodies
(C,F,I,L,O) and the DNAbinding dye DAPI (insets,
B,E,H,K,N). The immature
oocyte arrested at the dictyate
stage (A) contains a pair of
cytasters (B, arrows) tightly
associated with an intact
germinal vesicle (B, inset).
CKIα (C) is found only at the
plasma membrane as a
contiguous ring of staining and
is not detected in the GV
structure. In the metaphase II
arrested oocyte (D), CKIα (F) is
readily apparent on the spindle
apparatus (E) and the MVA (F,
arrowhead). Following
fertilization (G), the male and
female pronuclei develop in the
cytoplasm at 22 hours posthCG. Numerous cytoplasmic
microtubule organizing centers
(cytasters) and the second
meiotic midbody are detected
by anti-tubulin staining (H) at
this stage. CKIα (I) is not
detected in the cortex region as
noted in the unfertilized oocyte
(see A-C) but instead is
detected in both pronuclei,
staining the nucleoplasm in a
non-homogeneous distribution.
Neither the cytaster nor the
midbody structure is detected
by CKIα antibody staining following permeablization and fixation of these interphase cells. At first mitosis (J), the spindle (K) clearly has
associated CKIα (L). Following first cleavage (M), the midbody microtubules are readily visualized by tubulin antibodies (N). At this stage, CKIα
is generally distributed throughout the cytosol and appears to be concentrated at the nuclear envelope (O). Bar, 10 µm.
DISCUSSION
Here we have shown that a casein kinase I isoform is present
in mouse oocytes using immunoblotting, immunoprecipitation/kinase assays and immunofluorescence techniques.
Western blotting results using a CKIα-specific antibody
demonstrated that a single immunoreactive band is detected in
lysates derived from both mouse oocytes and a mouse cell line.
Based upon its observed SDS-PAGE mobility of 37 kDa, which
is very similar to the 37.5 kDa calculated molecular mass of
CKIα, the phosvitin kinase activity that immunoprecipitated
with the CKIα antibody and the previously defined specificity
of the antibody, this isoform is most likely CKIα (Brockman
et al., 1992; Gross et al., 1995; Zhang et al., 1996).
Dependent upon the specific developmental stage to which
the oocytes progress, there are both significant similarities and
fundamental differences in the subcellular distribution of the
kinase compared to that previously reported. Within meiosis I
stage oocytes, CKI’s association with the plasma membrane and
absence on the first meiotic spindle seemingly contradict earlier
findings which described an association with cytosolic vesicular
structures and the spindle apparatus in established cell lines
(Brockman et al., 1992). The kinase is not observed within the
germinal vesicle, unlike established cell lines where at least
some nuclear signal is always apparent (Brockman et al., 1992).
Such differences could be ascribed to the unique nature of the
germinal vesicle stage oocyte which is in a state of indefinite
arrest, awaiting specific signals to reenter the cell cycle. One
possibility is that CKI may associate with these structures in
order to help transduce the signals to reenter the cell cycle, or,
3088 S. D. Gross and others
Fig. 3. Microinjection of CKIα antibody into mature oocytes. (A, DIC) demonstrates that the kinase (C, CKI) strongly associates with the
second meiotic spindle and cytoplasmic cytasters (B, tubulin) but is not apparent on the condensed chromosomes as defined by the DNAbinding dye DAPI (B, inset). Microinjection of CKIα antibodies into living oocytes does not block chromosome segregation (E, DAPI; inset) or
second polar body formation (D, DIC) following parthenogenetic activation despite antibody localization to the spindle (F, CKI) as defined by
labeling with anti-tubulin antibodies (B and E). Bar, 10 µm.
essential for cell cycle progression in a number of oocyte
systems at the one-cell stage (Wormington, 1994).
These mouse oocyte studies were originally initiated to define
what role the kinase is performing when associated with mitotic
spindles. Studies in S. cerevisiae had linked particular isoforms of
the kinase to cytokinesis and chromosome segregation (Hoekstra
et al., 1991; Robinson et al., 1993). CKIα antibodies were
therefore microinjected at metaphase II-arrest and late G2 of the
first zygotic cell cycle to determine whether the kinase might be
involved in chromosome congression, segregation or cytokinesis.
Although no effects were observed, these results do not preclude
Percentage Oocytes Undergoing First
Cleavage
alternatively, this association with the plasma membrane may
actually prevent the kinase from interacting with and regulating
factors essential to further development, including nuclear
factors. In agreement with previous reports, CKIα is found on
both the spindle of metaphase II-arrested oocytes and zygotes
undergoing first cleavage and is readily detectable in the
pronuclei of 1-cell embryos (Brockman et al., 1992).
In metaphase II-arrested oocytes, CKIα was also observed to
associate with the MVA. This structure is thought to be involved
not only in defining oocyte polarity in metaphase II arrest but
also in spindle reorientation and polar body formation upon fertilization. Such functions correspond strongly with the cytokinetic defects associated with genetic lesions in the S. cerevisiae CKI homologues YCK1 and 2 (Robinson et al., 1993).
However, the exact nature of CKI’s role in cell division in any
system remains to be defined. Interestingly, evidence exists
linking phosphorylation, particularly via MAP kinase action, to
the regulation of these events (Verlhac et al., 1993). In light of
the fact that a number of other kinases are linked to MAP
kinase-associated signal transduction cascades in other systems,
it is likely that other kinases also play a role in the regulation
of these developmental events in the mouse oocyte. CKIα is at
the very least physically positioned to act in such a pathway.
Another important observation to note here is the nuclear
association of the kinase. CKI is not found in germinal vesicles
or the nuclei of newly-divided daughter cells of the two-cell
embryo. These structures are known to exhibit little, if any,
RNA and DNA synthesis activity. There exists evidence linking
CKI to the regulation of replication and transcription as well as
RNA processing, particularly the addition of poly(A) tails to
mRNA (Cegielska and Virshup, 1993; Dahmus, 1981; Hoekstra
et al., 1991; Murao et al., 1989, 1990; Rose and Jacob, 1979).
Potentially, this evidence could explain the observed differences
in nuclear localization. At least for DNA replication and mRNA
processing, these processes have been demonstrated to be
Mock injected
CKIα Ab injected
Control IgG injected
n=25
100 n=25
n=23
n=23
80
60
n=34
40
n=34
20
39 hours post-hCG
43 hours post-hCG
Fig. 4. Percentage of mouse zygotes undergoing in vitro cleavage
following microinjection of CKIα antibodies into pronucleate eggs.
Fertilized oocytes were microinjected with either buffer alone (mock)
or buffer containing non-specific rabbit IgG (IgG control) or buffer
with CKIα antibodies and then visually scored for progress through
to the two-cell stage at 39 and 43 hours post-hCG.
CKI in the mouse oocyte cell cycle 3089
Fig. 5. Microinjection of CKIα antibodies into
metaphase II-arrested oocytes blocks nuclear
importation of the kinase into the pronucleus
subsequent to parthenogenetic activation.
Metaphase II-arrested oocytes were microinjected
with control rabbit IgGs or CKIα antibodies,
parthenogenetically activated, and then fixed and
stained with CKIα antibodies. (A and C) Phase
contrast micrographs of oocytes injected with
control and CKIα antibodies, respectively. (B and
D) Representative of the signal for CKI. Note that
the oocyte injected with CKIα antibodies (D) does
not exhibit any detectable pronuclear signal in
contrast to the CKIα-immunodepleted IgGinjected oocyte (B). Bar, 10 µm.
the kinase from being involved in mitotic events. These CKIαantibodies are not particularly inhibitory (Gross et al., 1995) and
consequently, any sort of effect induced by microinjection of these
probes will likely be due to the disruption of critical proteinprotein interactions. Because the kinase is already associated with
the spindle apparatus at metaphase II arrest, it is conceivable that
the antibodies are sterically blocked from binding the kinase to
such an extent as to elicit a phenotype. Microinjection of CKIα
antibodies clearly does not have an effect on the association of the
kinase with spindles at metaphase II or first mitosis (Fig. 3A-C
and not shown) or on subsequent developmental events such as
chromosome congression, segregation and in the case of the latter,
cytokinesis. Alternatively, other CKI isoforms may have overlapping cell-division functions and compensate for the loss of CKIα.
Microinjection studies using antibodies to CKIα demonstrate
that there was a temporal component to the developing oocyte’s
sensitivity to addition of this probe. If CKIα antibodies were
introduced into oocytes immediately after fertilization at sperm
incorporation, progression to first cleavage was significantly
impeded. Introduction of these same antibody probes at 8 hours
post-fertilization had no effect on subsequent progression to the
two-cell stage. In addition, the observed developmental block
could very well have been more dramatic if it were not for the
inherent developmental variation in the population of oocytes
that were microinjected. Such a variation is supported by the
fact that a number of the oocytes receiving CKIα antibodies at
sperm incorporation were not blocked in development suggesting that the critical temporal window had been missed.
Within this temporal window, the kinase becomes associated
with discrete non-nucleolar pronuclear structures. Microinjection of CKIα antibodies into oocytes that are subsequently
activated completely blocks CKIα from relocalizing to these
intra-nuclear sites most likely by preventing the kinase’s
nuclear import. The complete absence of nuclear signal for
CKIα in these microinjected oocytes strongly supports this
interpretation (Fig. 5D). The observed block in proper and
timely cell cycle progression correlates strongly with the
kinase’s ability to interact with these nuclear structures. As
noted previously, the nuclear localization of Hrr25p and Yck3p
is essential for their function in the regulation of G1 pro-
gression, DNA replication and repair (Ho et al., 1997; Wang et
al., 1996). The temporal window of sensitivity described here
is positioned immediately prior to the onset of DNA synthesis.
Interestingly, a report by Santos et al. (1996) describes the relocalization of the Drosophila CKIα in early development from
the cytosol to the nucleus in response to γ-irradiation, a
treatment known to induce double-stranded breaks in DNA.
Moreover, recent evidence strongly indicates that the regulation of DNA synthesis and double-stranded break repair
employ some of the same regulatory pathways suggesting that
CKIα could be involved in these processes (Weaver, 1996), a
likelihood already established for the kinase homologues in
yeast (Dhillon and Hoekstra, 1994; Hoekstra et al., 1991).
While the phenotypes associated with the yeast CKI homologues demonstrate that they are involved in the regulation of a
number of critical cellular events related to cell cycle regulation, few data exist linking higher eukaryotic isoforms of CKI
to these events. CKIδ and CKIε can each complement HRR25
function (Fish et al., 1995) and CKIγ3 partially complements
YCK2 (Zhai et al., 1995) suggesting that these isoforms perform
parallel functional roles. While this evidence is compelling, γ3
complementation was only partial and required that the gene for
this CKI isoform be introduced on a high copy number plasmid
(Zhai et al., 1995). In addition, a number of studies have demonstrated that CKI may be involved in the regulation of both G1/S
transit and cytokinesis in more complex systems. CKIα exhibits
a cell cycle-dependent subcellular localization that includes an
association with the mitotic spindle suggesting a potential role
in cell division similar to that genetically linked to the YCK1
and 2 isoforms. In addition, CKI has been purified from whole
cell lysates based upon its ability to phosphorylate both SV40
large T antigen (Cegielska et al., 1994; Cegielska and Virshup,
1993) and p53 (Milne et al., 1992). In particular, CKIα, when
introduced into an in vitro SV40 DNA replication reaction, will
phosphorylate SV40 large T on serine residue 123 effecting a
significant reduction in this viral protein’s replicative activity
(Cegielska et al., 1994; Cegielska and Virshup, 1993; Grasser
et al., 1988; Tuazon and Traugh, 1991). It has also been shown
that a CKI isoform phosphorylates p53 on serine residue 9
within the transcriptional activation domain (Milne et al., 1992).
3090 S. D. Gross and others
Although this residue is phosphorylated in vivo, no modulation
of p53 activity has yet been associated with modification of this
amino acid (Milne et al., 1992) and recent evidence suggests
that this particular modification has no effect on the transcriptional activity of p53 in vivo (Fuchs et al., 1995).
The results outlined in this study are, to our knowledge, the
first in vivo evidence indicating that higher eukaryotic isoforms
of CKI may be involved in the regulation of cell cycle progression and, in particular, early mammalian development.
The work was funded by the National institutes of Health to G.S.
(USDA 93-01686 and NIH HD12913) and R.A.A. (NIH GM51968
and GM38906). We than Drs L. Hewiston and S. Zoran for their
helpful discussions and figure preparation.
REFERENCES
Brockman, J. L., Gross, S. D., Sussman, M. R. and Anderson, R. A. (1992).
Cell cycle-dependent localization of casein kinase I to mitotic spindles. Proc.
Nat. Acad. Sci. USA 89, 9454-9458.
Cegielska, A., Moarefi, I., Fanning, E. and Virshup, D. M. (1994). T-antigen
kinase inhibits simian virus 40 DNA replication by phosphorylation of intact
T antigen on serines 120 and 123. J. Virol. 68, 269-275.
Cegielska, A. and Virshup, D. M. (1993). Control of simian virus 40 DNA
replication by the HeLa cell nuclear kinase casein kinase I. Mol. Cell. Biol.
13, 1202-1211.
Dahmus, M. E. (1981). Phosphorylation of eukaryotic DNA-dependent RNA
polymerase. J. Biol. Chem. 256, 3332-3339.
DeMaggio, A. J., Lindberg, R. A., Hunter, T. and Hoekstra, M. F. (1992).
The budding yeast HRR25 gene product is a casein kinase I isoform. Proc.
Nat. Acad. Sci. USA 89, 7008-7012.
Dhillon, N. and Hoekstra, M. F. (1994). Characterization of two protein
kinases from Schizosaccharomyces pombe involved in the regulation of DNA
repair. EMBO J. 13, 2777-2788.
Fish, K. J., Cegielska, A., Getman, M. E., Landes, G. M. and Virshup, D. M.
(1995). Isolation and characterization of human casein kinase I epsilon
(CKI), a novel member of the CKI gene family. J. Biol. Chem. 270, 1487514883.
Fuchs, B., O’Connor, D. Fallis, L., Scheidtmann, K. H. and Lu, X. (1995). p53
phosphorylation mutants retain transcription activity. Oncogene 10, 789-793.
Gavin, A. C. and Schorderetslatkine, J. (1997). Ribosomal S6 kinase p90
(rsk) and mRNA cap-binding protein eIF-4E phosphorylations correlate with
MAP kinase activation during meiotic reinitiation of mouse oocytes. Mol.
Reprod. Dev. 46, 383-391.
Grana, X. and Reddy, E. P. (1995). Cell cycle control in mammalian cells: role
of cyclins, cyclin dependent kinases (CDKs), growth suppressor genes and
cyclin-dependent kinase inhibitors (CKIs). Oncogene 211-219.
Grasser, F. A., Scheidtmann, K. H., Tuazon, P. T., Traugh, J. A. and Walter,
G. (1988). In vitro phosphorylation of SV40 large T antigen. Virology 165,
13-22.
Graves, P. R., Haas, D. W., Hagedorn, C. H., DePaoli-Roach, A. A. and
Roach, P. J. (1993). Molecular cloning, expression and characterization of a
49-kilodalton casein kinase I isoform from rat testis. J. Biol. Chem. 268,
6394-63401.
Gross, S. D., Hoffman, D. P., Fisette, P. L., Baas, P. and Anderson, R. A.
(1995). A phosphatidylinositol 4,5-bisphosphate-sensitive casein kinase I
alpha associates with synaptic vesicles and phosphorylates a subset of vesicle
proteins. J. Cell Biol. 130, 711-724.
Ho, Y., Mason, S., Kobayashi, R., Hoekstra, M. and Andrews, B. (1997).
Role of the casein kinase I isoform, Hrr25 and the cell cycle-regulatory
transcription factor, SBF, in the transcriptional response to DNA damage in
Saccharomyces cerevisiae. Proc. Nat. Acad. Sci. USA 94, 581-586.
Hoekstra, M. F., Liskay, R. M., Ou, A. C., DeMaggio, A. J., Burbee, D. G.
and Heffron, F. (1991). HRR25, a putative protein kinase from budding
yeast: association with repair of damaged DNA. Science 253, 1031-1034.
Kaufman, M. H. (1983). Early Mammalian Development: Parthenogenetic
Studies. Cambridge, UK: Cambridge University Press.
Kearney, P. H., Ebert, M. and Kuret, J. (1994). Molecular cloning and
sequence analysis of two novel fission yeast casein kinase-1 isoforms.
Biochem. Biophys. Res. Commun. 203, 231-236.
Machlachlan, T. K., Sang, N. and Giordano, A. (1995). Cyclins, cyclindependent kinase and cdk inhibitors: implications in cell cycle control and
cancer. Crit. Rev. Euk. Gene Expres. 5, 127-156.
Milne, D. M., Palmer, R. H., Campbell, D. G. and Meek, D. W. (1992).
Phosphorylation of the p53 tumour-suppressor protein at three N-terminal
sites by a novel casein kinase I-like enzyme. Oncogene 7, 1361-1369.
Murao, S., Collart, F. R. and Huberman, E. (1989). A protein containing the
cystic fibrosis antigen is an inhibitor of protein kinases. J. Biol. Chem. 264,
8356-8360.
Murao, S., Collart, F. and Huberman, E. (1990). A protein complex
expressed during terminal differentiation of monomyelocytic cells is an
inhibitor of cell growth. Cell Growth Differ. 1, 447-454.
Nigg, E. A. (1996). Cyclin-dependent kinase 7: at the cross-roads of
transcription, DNA repair and cycle cycle control? Curr. Opin. Cell Biol. 8,
312-317.
Pines, J. (1994). Protein kinases and cell cycle control. Semin. Cell Biol. 5, 399408.
Robinson, L. C., Hubbard, E. J., Graves, P. R., DePaoli-Roach, A. A.,
Roach, P. J., Kung, C., Haas, D. W., Hagedorn, C. H., Goebl, M.,
Culbertson, M. R., et al. (1992). Yeast casein kinase I homologues: an
essential gene pair. Proc. Nat. Acad. Sci. USA 89, 28-32.
Robinson, L. C., Menold, M. M., Garrett, S. and Culbertson, M. R. (1993).
Casein kinase I-like protein kinases encoded by YCK1 and YCK2 are
required for yeast morphogenesis. Mol. Cell. Biol. 13, 2870-2881.
Rose, K. M. and Jacob, S. T. (1979). Phosphorylation of nuclear poly(A)
polymerase. J. Biol. Chem. 254, 10256-10261.
Rowles, J., Slaughter, C., Moomaw, C., Hsu, J. and Cobb, M. H. (1991).
Purification of casein kinase I and isolation of cDNAs encoding multiple
casein kinase I-like enzymes. Proc. Nat. Acad. Sci. USA 88, 9548-9552.
Santos, J. A., Logarhino, E., Tapia, C., Allende, C. C., Allende, J. E. and
Sunkel, C. E. (1996). The casein kinase 1α gene of Drosophila
melanogaster is developmentally regulated and the kinase activity of the
protein induced by DNA damage. J. Cell Sci. 109, 1847-1856.
Simerly, C., Balczon, R., Brinkley, B. R. and Schatten, G. (1990).
Microinjected kinetochore antibodies interfere with chromosome movement
in meiotic and mitotic mouse oocytes. J. Cell Biol. 111, 1491-1504.
Simerly, C. and Schatten, G. (1993). Techniques for localization of specific
molecules in oocytes and embyros. Meth. Enzymol. 225, 516-553.
Stefanovic, D. and Maller J. L. (1988). Post-transcriptional regulation by
insulin of Xenopus ribosomal protein S6 kinase. Exp. Cell Res. 179, 104-114.
Tuazon, P. T. and Traugh, J. A. (1991). Casein kinase I and II--multipotential
serine protein kinases: structure, function and regulation. Advan. Second
Mess. Phosphoprot. Res. 23, 123-164.
Vancura, A., Sessler, A., Leichus, B. and Kuret, J. (1994). A prenylation
motif is required for plasma membrane localization and biochemical function
of casein kinase I in budding yeast. J. Biol. Chem. 269, 19271-19278.
Verlhac, M.-H., Pennart, H., Maro, B., Cobb, M. H. and Clarke, H. J.
(1993). MAP kinase becomes stably activated at metaphase and is associated
with microtubule-organizing centers during meiotic maturation of mouse
oocytes. Dev. Biol. 158, 330-340.
Wang, P.-C., Vancura, A., Mitcheson, T. G. M. and Kuret, J. (1992). Two
genes in Saccharomyces cerevisiae encode a membrane-bound form of
casein kinase-1. Mol. Biol. Cell 3, 275-286.
Wang, X., Hoekstra, M. F., DeMaggio, A. J., Dhillon, N., Vancura, A.,
Kuret, J., Johnston, G. C. and Singer, R. A. (1996). Prenylated isoforms of
yeast casein kinase I, including the novel Yck3p, suppress the gcs1 blockage
of cell proliferation from stationary phase. Mol. Cell. Biol. 16, 5375-5385.
Weaver, D. T. (1996). Regulation and repair of double-stranded breaks. Crit.
Rev. Euk. Gene Express. 6, 345-375.
Wormington, M. (1994). Unmasking the role of the 3’ UTR in the cytoplasmic
polyadenylation and translational regulation of maternal mRNAs. BioEssays
16, 533-535.
Zhai, L., Graves, P. R., Robinson, L. C., Italiano, M., Culbertson, M. R.,
Rowles, J., Cobb, M. H., DePaoli-Roach, A. A. and Roach, P. J. (1995).
Casein kinase I gamma subfamily. Molecular cloning, expression and
characterization of three mammalian isoforms and complementation of
defects in the Saccharomyces cerevisiae YCK genes. J. Biol. Chem. 270,
12717-12724.
Zhang, J., Gross, S. D., Schroeder, M. D. and Anderson, R. A. (1996).
Casein kinase Iα and αL: Alternative splicing-generated kinases exhibit
different catalytic properties. Biochemistry 35, 16319-16327.
(Accepted 19 September 1997)