(Received for publication, August 16, 1994; and in revised form, January 13, 1995)
From the
Earlier work demonstrated that cyclins A1, B1, and B2 are not
associated with Cdk2 from unfertilized Xenopus eggs. As a
potential Cdk2 partner during meiosis, a cyclin E homolog was cloned
from a Xenopus oocyte cDNA library and found to be 60%
identical at the amino acid level to human cyclin E. Cyclin E1 protein
was detected in resting oocytes, and the level increased severalfold in
meiosis II, concomitant with the appearance of forms with decreased
electrophoretic mobility. During oocyte maturation, the patterns of
cyclin E1-associated kinase activity and Cdk2 activity were identical,
with activity low until after germinal vesicle breakdown, peaking
during meiosis II. Cyclin E1 complexes immunoprecipitated from
unfertilized Xenopus eggs contained Cdk2 but not Cdc2. In
cycling egg extracts Cdk2-cyclin E1-associated kinase activity
oscillated, but the level of cyclin E1 protein and its association with
Cdk2 did not vary appreciably; complex activity appeared to be
regulated neither by the synthesis and destruction of the cyclin
subunit nor by association/disassociation of the two subunits. During
the early cleavage divisions in embryos, cyclin E1 and Cdk2 remained
associated. The data indicate that the Cdk2-cyclin E complex functions
during meiotic and embryonic cell cycles in addition to performing its
established role during G in somatic cells.
Many proteins involved in cell cycle control are evolutionarily
conserved from yeast to man. A number of human cyclin-dependent kinase
(cdk) and cyclin gene products have been identified that complement
cell cycle defects present in budding and fission yeast
mutants(1, 2, 3, 4, 5, 6) .
Cdc2, Cdk2, and cyclin A and B homologs have also been cloned from Xenopus
laevis(7, 8, 9, 10) . In fact,
three cell cycle regulators, maturation-promoting factor (MPF), ()cytostatic factor (CSF), and Cdk2 were discovered in Xenopus oocytes and eggs(10, 11) . MPF
consists of a complex of Cdc2 and cyclin B, and its activation
regulates entry into both meiosis and
mitosis(12, 13, 14) . CSF maintains MPF
activity and is responsible for the unique metaphase arrest in meiosis
II characteristic of vertebrate unfertilized eggs(11) . CSF
activity requires the c-mos proto-oncogene
product(15, 16) and other components(17) ,
including Cdk2(18) .
So far Cdk2 has been most thoroughly
characterized in somatic tissue culture cells. Cdk2 associates with A-
and E-type cyclins during mitotic cell
cycles(2, 19, 20, 21, 22, 23, 24, 25, 26) .
Complexes of Cdk2 with cyclin A are activated at the beginning of S
phase (27) and may be necessary for DNA
replication(24, 28, 29) . When associated
with Cdc2, cyclin A also has a G/M function in somatic cell
cycles (29) and may participate in S/M checkpoint control in
embryonic cycles(30) . Cdk2 associates in vitro with
the D1, D2, and D3 cyclins to variable extents; in mammalian cells,
however, kinase-active complexes of D-type cyclins have been
demonstrated with Cdk4 and Cdk6, but not with
Cdk2(31, 32) . Moreover, the D1 and D2 cyclins have
proven to be undetectable in Xenopus eggs or embryos prior to
the midblastula transition.(
)(
)Another Cdk2
cyclin partner is cyclin E. In tissue culture cells, Cdk2-cyclin E
complexes assemble in the middle of G
, with associated
kinase activity peaking in late G
and extending into early
S phase(20, 22) . Overexpression of human cyclin E in
mammalian fibroblasts shortens the duration of the G
period, suggesting that cyclin E may be rate-limiting for G
progression(33, 34) . Additionally, inhibition
of Cdk2-cyclin E complex activity by transforming growth factor-
in mammalian cells blocks G
progression(35) . A
G
block is also observed when a dominant-negative form of
Cdk2 is expressed in cells(36) . Cdk2-cyclin E complexes have
been implicated in the regulation of late G
events that
allow G
exit, including phosphorylation of p107, a protein
related to the retinoblastoma gene
product(23, 35, 37, 38, 39, 40, 41) .
When considering additional functions for Cdk2, it should be noted that meiotic, embryonic, and somatic cell cycles differ fundamentally in the relationship coupling genomic replication to cellular division. Meiotic cell cycles involve one round of DNA replication followed by two successive rounds of cellular division resulting in the formation of haploid reproductive cells, whereas during somatic cell cycles DNA replication is faithfully followed by mitosis and cytokinesis resulting in two diploid daughter cells(42) . In Xenopus embryonic cycles mitosis is not dependent on successful DNA replication until the midblastula transition(43, 44) . Despite the difference in the coupling of events in the cell cycle, many proteins, such as MPF, CDC7 in yeast, and Cdk2 play roles in meiosis as well as mitosis(12, 18, 45, 46) . A role for Cdk2 in meiosis is not unexpected because the synthesis of Cdk2 increases during Xenopus oocyte maturation(47) . Differences in cell cycle control may be partially defined by the different constituents and timing of various cdk complexes(48) . For instance, it appears that the A cyclin- and B cyclin-associated cdks are active at different times in Xenopus egg extracts(8, 30) . In vitro, different cyclin-cdk complexes may be targeted to specific substrates(39) . In somatic cells cyclin A and cyclin E, but not cyclin B, are found in complexes with Cdk2, E2F, and p107 at discrete times in the cell cycle, and these complexes can be sequestered by the transforming proteins of DNA tumor viruses(27, 49) . However, E2F appears not to form these types of complexes in the early embryonic cell cycles of Xenopus(50) , suggesting that Cdk2 has functions in early embryonic cell cycles distinct from those in somatic cycles.
In Xenopus meiotic cell cycles, the A, B1, or B2 cyclins do
not form complexes in vivo with
Cdk2(8, 47, 51) . The present study was
initially designed to resolve whether cyclin E could be the meiotic
partner of Xenopus Cdk2. A homolog to human cyclin E was
cloned from a Xenopus oocyte library and designated cyclin E1.
During oocyte maturation the level of cyclin E1 protein and associated
kinase activity was found to increase significantly, both peaking in
meiosis II. The pattern of cyclin E1-associated kinase activity
paralleled that of Cdk2. Furthermore, kinase-active cyclin E1
immunocomplexes contained Cdk2 but not Cdc2. The behavior of cyclin E1
was also examined in Xenopus egg extracts (CSF extracts) that
carry out cell cycle transitions in vitro. Such extracts begin
in metaphase with high total H1 kinase activity, progress into
interphase with low H1 kinase activity and DNA replication, and then
cycle back into M phase with chromatin condensation, nuclear envelope
breakdown, and high H1 kinase activity. During an extract cell cycle
cyclin E1-associated kinase activity oscillated but the level of cyclin
E1 protein was stable and association with Cdk2 did not vary. The
cyclin E1-Cdk2 complex identified in eggs remained present during the
early cleavage stages of embryos. This maternal form of cyclin E
eventually disappeared between stages 8 and 10.5, coincident with the
onset of zygotic transcription and the first appearance of
G-phase cells(52) .
Metaphase II-arrested CSF
extracts were prepared from unfertilized Xenopus eggs as
described (53) except that leupeptin was omitted from all
buffers. Generally, extracts were supplemented at time 0 with both 400
µM CaCl and demembranated sperm nuclei
(100-200/µl). When indicated, extracts were treated at time 0
with emetine (100 µM) or aphidicolin/DNA (50 µg/ml
aphidicolin and 3000 nuclei/µl).
For embryos, eggs were
fertilized in vitro, dejellied in 2% cysteine, pH 7.8, washed
in 0.1 MMR (10 mM NaCl, 0.2 mM KCl, 0.1
mM MgSO
, 0.2 mM CaCl
, and 0.5
mM Hepes, pH 7.8), and then cultured at room temperature in
0.1
MMR. The embryos were staged according to the table of
Nieuwkoop and Faber(54) . Embryos were collected at the
indicated stages, frozen on dry ice, and stored at -80 °C.
For analysis, the embryos were homogenized in 10 volumes of EB plus 1
µM microcystin, centrifuged for 5 min in a
microcentrifuge, and the supernatants used for immunoblotting and
immunoprecipitation.
Antiserum was raised in rabbits against a synthetic peptide of the C terminus of Xenopus Cdk2 and affinity purified on a Cdk2 peptide column as described previously (47) with the following exceptions: the Cdk2 peptide differed at the N-terminal position, with a cysteine replacing the original threonine, and coupling of the peptide was to thiopropyl-Sepharose beads (Pharmacia). Antiserum to Cdc2 was generated in rabbits against a synthetic peptide of the C terminus of Xenopus Cdc2 and affinity purified using a Cdc2 peptide column. Antiserum was raised in sheep against full-length Xenopus cyclins A1, B1, and B2(63) . The B1 and B2 antisera were used without further purification whereas the cyclin A1 antiserum was affinity-purified using a column of Sepharose 4B coupled to recombinant cyclin A1.
For
immunoblot analysis, the amount of oocyte extract used per gel lane
represented 70% of an oocyte (40 µg of protein), the amount of CSF
extract analyzed was 0.5 µl, and the amount of egg or embryo
extract represented the equivalent of one egg or embryo. Samples were
resolved by electrophoresis on 10% polyacrylamide gels for cyclin
detection and on 12.5% polyacrylamide gels for Cdk2 and Cdc2 detection (62) . Proteins were transferred to nitrocellulose and the
membranes rinsed in 1 PBS and blocked by incubation in blotto
for 2 h at room temperature. Primary antibody incubations were
overnight at 4 °C in blotto. For detection of cyclin E1, the
blot-purified antibody described above was diluted 1:1 with blotto.
Detection of cyclin E1 was blocked by preincubation of the
blot-purified antibody with cyclin E1 (15 µg of E/ml of antibody).
Cdk2 was detected using affinity-purified antibody and blocked by
preincubation of the antibody with immunizing peptide.
Affinity-purified antibodies were used for the detection of Cdc2 and
cyclin A1. The blots were incubated with the appropriate horseradish
peroxidase-conjugated secondary antibody (Jackson Immunochemicals, West
Grove PA) at 1:10,000 and visualized by ECL (Amersham, Arlington
Heights IL).
Endogenous cyclin E1 was quantitated by immunoblot
analysis. Blot-purified antibody precleared of His-reactive
antibodies was used to allow direct comparison of the signal generated
by His
-tagged recombinant cyclin E1 to that of endogenous
E1. The depleted antibody was generated by incubation of the
blot-purified antibody to cyclin E1 with nitrocellulose strips of
His
-tagged cyclin B2. The signal generated by cyclin E1 in
extract samples was compared to that generated by known amounts of
purified soluble recombinant cyclin E1 using densitometric scanning of
the immunoblot-exposed film with ImageQuant software (Molecular
Dynamics, Sunnyvale CA).
Immunoprecipitates were incubated with 25 µl of a buffer
containing 20 mM Hepes, pH 7.5, 15 mM MgCl, 5 mM EGTA, 1 mM DTT, 0.2 mg/ml
bovine serum albumin, 0.5 mg/ml histone H1, and 200 µM [
-
P]ATP (2 cpm/fmol). Samples were
incubated at 25 °C for 20 min and the reaction stopped by the
addition of 25 µl of 2
sample buffer(62) . For
assays of total histone H1 kinase activity, 3 µl of extract was
diluted 10-fold in buffer (80 mM
-glycerophosphate, 15
mM MgCl
, and 5 mM EGTA) and 3 µl then
assayed in duplicate. To each aliquot was added 12 µl of a kinase
mixture (20 mM Hepes, pH 7.5, 15 mM MgCl
,
5 mM EGTA, 1 mM DTT, 0.2 mg/ml bovine serum albumin,
15 µg/ml heat stable inhibitor of protein kinase A, 0.5 mg/ml
histone H1, and 200 µM [
-
P]ATP
(5 cpm/fmol)). Samples were incubated at 30 °C for 15 min and the
reaction terminated by the addition of 5 µl of 5
sample
buffer (62) . Immunoprecipitated and total extract samples were
boiled for 3 min, electrophoresed on 12.5% polyacrylamide gels, and
histone H1 phosphorylation determined by Cerenkov counting of the
excised H1 band.
Prior to treatment with
phosphatase, both sets of immunoprecipitates were washed twice in a
phosphatase buffer (50 mM Tris-HCl, pH 7.4, 30 mM NaCl, 1 mM EDTA, 2 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, or 200 µM Pefabloc,
and 10 µg/ml each aprotinin, leupeptin, chymostatin, and
pepstatin). Endogenous cyclin E1 immunoprecipitates were incubated for
30 min at 23 °C with either buffer, recombinant X. laevis PP1-1, purified PP2A (bovine cardiac PP2A
catalytic subunit, kindly provided by Dr. M. Mumby, Southwestern
Medical Center, Dallas, TX), or each phosphatase inactivated by
coincubation with microcystin (8 µM). Radiolabeled cyclin
E1 was incubated under the same conditions with buffer, PP1-
1, or
PP1-
1 preincubated with 10 µM microcystin. The
endogenous and [
-
P]ATP-labeled cyclin E1
immunoprecipitates were boiled in sample buffer with 10 mMN-ethylmaleimide or 5%
-mercaptoethanol,
respectively, and fractionated on 10% polyacrylamide gels. Endogenous
cyclin E1 was visualized by immunoblotting with blot-purified goat
antibody to cyclin E1, whereas radiolabeled cyclin E1 was visualized by
autoradiography.
Figure 1: Nucleotide sequence of X. laevis cyclin E1 and the predicted amino acid sequence of its gene product. The region of the open reading frame is shown. The methionine codon selected as the initiator is appropriately positioned relative to an upstream purine residue, in keeping with the Kozak consensus sequence(89) , and is downstream from an in-frame stop codon, marked by an underline. The termination codon is indicated by an asterisk. Nucleotide and amino acid numbers are indicated on the right. The single-letter code is used for the amino acids. The sequence has been deposited in GenBank under accession number L23857.
Figure 2: Panel A, alignment of Xenopus cyclin E1 with human cyclin E. Optimal alignment did not require the insertion of gaps. Dashes indicate identical amino acids. The cyclin box domain is highlighted in bold. Panel B, alignment of Xenopus cyclin E1 with Xenopus cyclins A1, B1, and B2. Residues common to all four sequences are highlighted, and dashes indicate gaps. The cyclin box domain is enclosed. The human cyclin E sequence is taken from Lew et al.(3) .
Expression of the cyclin E1 gene was assessed by Northern blot analysis (Fig. 3). Hybridization with a probe derived from the coding
region of the Xenopus cDNA clone revealed a single major
species of approximately 2.3 kilobase pairs. The cyclin E1 mRNA was detected in oocytes, eggs, and early embryos and
therefore represents a maternal mRNA species. This mRNA species
remained detectable through the onset of zygotic transcription (stage
8) and gastrulation (stage 10), but was declining by stage 13 (data not
shown). A mRNA of similar molecular weight was found in the Xenopus tissue culture line (XTC) after synchronization in G/S
phase by thymidine treatment (data not shown). Cyclin E1 mRNA
levels did not vary significantly during early development when
adjusted for the level of c-src mRNA, shown in the bottom
panel. The mRNA for cyclin E1 was not found to be deadenylated
upon fertilization (data not shown), in contrast to the case for other
transcripts such as cdk2 mRNA (10) .
Figure 3: The expression of cyclin E1 mRNA during Xenopus development. For Northern analysis, total RNA was isolated from oocytes, unfertilized eggs, and staged embryos, fractionated by denaturing agarose gel electrophoresis, and transferred to nitrocellulose. Panel A, the blot was probed with the coding region of the Xenopus cyclin E1 cDNA. Panel B, the same blot was re-probed with the Xenopus src-1 cDNA(59) . The level of src mRNA remains fairly constant throughout early Xenopus development and thus serves as a control for RNA loading(58) . Positions of migration of the RNA molecular weight markers are indicated at the left; the cyclin E1 and src-1 transcripts are indicated at the right by arrows.
Figure 4:
Specificity of the antibody to Xenopus cyclin E1 and analysis of cyclin E1 protein and associated kinase
activity during progesterone-induced oocyte maturation. Panel
A, CSF metaphase-arrested extract from Xenopus eggs was
immunoblotted with blot-purified antiserum to cyclin E1 or the same
antiserum blocked by prior incubation with recombinant cyclin E1. Panels B and C, cyclin E1 and Cdk2 during oocyte
maturation. Time 0 represents stage VI oocytes to which no progesterone
was added. Time after progesterone addition is indicated in
hours:minutes and 50% GVBD is indicated by the arrow.
Immunoblots were probed with blot-purified immune serum to cyclin E1 (panel B) or affinity-purified immune serum to Cdk2 (panel
C). Panel D, cyclin E1-associated kinase activity during
oocyte maturation. Samples of 10 oocytes were assayed for H1 kinase
activity in cyclin E1 immunoprecipitates (filled circles) or
Cdc2 immunoprecipitates (open squares). These samples were
identical to those used for immunodetection in panels B and C. The amount of P incorporated during a 20-min
kinase assay was corrected for the activity in immunoprecipitations
blocked by prior incubation with the antigen. The arrow indicates 50% GVBD at 3 h 20 min.
When endogenous cyclin E1 was immunoprecipitated from CSF extracts made from unfertilized Xenopus eggs, kinase activity toward histone H1 was readily detected; this activity was 10-fold greater than the activity present when either preimmune serum, or immune serum blocked by preincubation with recombinant cyclin E1, were used ( Fig. 7and data not shown). The activity was substantial, approximately 10-20% of that immunoprecipitated with antibodies to Cdc2 or the B1 and B2 cyclins (Fig. 4D, 5A, and 7).
Figure 7:
Cyclin-Cdk complexes in a CSF
metaphase-arrested extract. Immunoprecipitations were performed using
beads, goat preimmune serum, goat affinity-purified serum to cyclin E1,
sheep serum to cyclin A1, and sheep sera to cyclin B1 and cyclin B2.
Supernatant and pellet fractions were immunoblotted and kinase assays
performed on duplicate pellet fractions. The kinase activity of the
immunoprecipitated complexes (expressed as pmol of P
incorporated into histone H1 during a 20-min kinase assay) were as
follows: beads, 1.7; preimmune, 4.3; cyclin E1, 57; cyclin A1, 11;
cyclins B1/B2, 401. Panel A, supernatant and pellet fractions
immunoblotted for Cdk2. Two forms of Cdk2 were resolved. Panel
B, supernatant and pellet fractions immunoblotted for
Cdc2.
Cyclin E1-associated kinase activity was assessed during the same time course. The patterns of Cdc2 and cyclin E1-associated kinase activities are shown in Fig. 4D. Cdc2 activity was low in stage VI oocytes, rose to peak at GVBD, approximately 3 h, declined during the transition between meiosis I and II, and then rose again during meiosis II. Cyclin E1-associated kinase activity was distinct from that of Cdc2. There was very low activity until after GVBD and then activity gradually increased and remained elevated during meiosis II. The profile of kinase activity associated with cyclin E1 was similar to that reported previously for Cdk2(47) .
Figure 5:
Panel A, time course of cyclin
E1-associated kinase activity in a CSF extract released from metaphase
arrest by the addition of Ca. Samples from a CSF
extract were assayed for H1 kinase activity in cyclin E1
immunoprecipitates (filled circles) or Cdc2 immunoprecipitates (open squares). The amount of
P incorporated
during a 20-min kinase assay was corrected for the activity in
immunoprecipitations blocked by prior incubation with antigen. Panel B, time courses of cyclin E1-associated H1 kinase
activity in control (closed circles), emetine-treated (open squares), and aphidicolin + DNA-treated (open
triangles) CSF extracts. Panel C, immunoblot analysis of
cyclin E1 in control and emetine-treated CSF extracts. i, the
extract was released from metaphase arrest with Ca
and samples taken at the indicated times for immunoblot analysis; arrows indicate two prominent forms of cyclin E1. ii,
the extract was treated with the protein synthesis inhibitor emetine
prior to the addition of Ca
. Samples in the far right lanes curve upward due to a gel
artifact.
The activity during interphase, although
lower than in M phase, was still substantially higher than in blocked
control immunoprecipitations and may have functional significance. As
shown by Suc-1 depletion of cdks or specific immunodepletion of Cdk2,
DNA replication in the Xenopus extract system requires Cdk2
activity(51, 76) . Addition of Cdk2-cyclin E1 or
Cdk2-cyclin A1 complexes to depleted extracts restores DNA synthesis. ()The inhibition of DNA replication resulting from addition
of the cdk inhibitor, p21, can be prevented by the addition of cyclin
E1 or cyclin A1(77) . The interphase level of cyclin E1/Cdk2
activity, therefore, is likely to play a role in DNA synthesis.
Although the level of cyclin E1 did not vary
substantially, the electrophoretic mobility of cyclin E1 species did
vary. In the standard extract, before the addition of
Ca, a slower mobility form predominated; during the
intermediate time points, the majority of cyclin E1 had a faster
electrophoretic mobility; as the extract progressed into M phase there
was reaccumulation of the slower mobility forms (Fig. 5C, i). In an emetine-treated extract,
there was no reaccumulation of the slowest mobility form of cyclin E1 (Fig. 5C, ii) and there was a steady decline rather
than a reactivation of cyclin E1-associated kinase activity (Fig. 5B). The failure to reactivate cyclin
E1-associated kinase activity, despite the continued presence of cyclin
E1, might reflect the necessity for synthesis of some positive
regulator or titration of an inhibitor by other cyclin complexes. As
well, there may be a role for newly synthesized cyclin E1 in the
formation of active complexes; synthesis of cyclin E1 was detected in
control extracts by metabolic labeling with
[
S]methionine and immunoprecipitation of cyclin
E1 (data not shown).
It was previously reported that Cdk2, unlike
Cdc2, is activated in emetine-treated oscillating
extracts(47) ; cyclin E1-associated kinase activity also
increased in such extracts, although more gradually than in control
extracts (data not shown). The oscillating Murray extracts used in
those experiments, however, were prepared differently from the CSF
extracts discussed above. Murray extracts are prepared from eggs
released from metaphase arrest for 30 min before crushing; only then
were these extracts treated with the protein synthesis inhibitor. In
the CSF extracts emetine was added prior to the addition of
Ca to release metaphase arrest. One possibility is
that the signal for reactivation of cyclin E1 complexes, which requires
synthesis of some component, has already been established by 30 min
into the cell cycle. This might permit activation to occur in protein
synthesis-inhibited Murray extracts but not in CSF extracts.
The
different forms of cyclin E1 detected by immunoblotting could reflect
post-translational modification. As phosphorylation is a likely
modification, cyclin E1 immunoprecipitates from a CSF
metaphase-arrested extract were treated with the serine/threonine
protein phosphatases PP1 and PP2A (Fig. 6A). Although
the reaction did proceed to completion, a downward shift in cyclin E1
electrophoretic mobility was detected. When immunoprecipitates were
treated with the phosphatases inactivated by microcystin no downshift
occurred. When recombinant cyclin E1 was added to a CSF extract, there
was radiolabeling of the protein with
[-
P]ATP. This signal was largely removed by
treatment with PP1 but not by treatment with microcystin-inactivated
PP1 (Fig. 6B). Cyclin E1 is, therefore, a
phosphoprotein. The significance of this phosphorylation is not known,
but the phosphorylation may be an autophosphorylation reaction that
reflects the relative activity of the kinase complex.
Figure 6:
Phosphatase treatment of cyclin E1
immunoprecipitates. Panel A, cyclin E1 was immunoprecipitated
from a CSF metaphase-arrested extract using immune rabbit serum and the
immune complexes treated with buffer alone (lane a), PP1
catalytic subunit (lane b), PP1 and 8 µM microcystin (lane c), PP2A catalytic subunit (lane
d), or PP2A and 8 µM microcystin (lane e).
The treated immunoprecipitates were resolved by SDS-polyacrylamide gel
electrophoresis, transferred to nitrocellulose, and immunoblotted using
the blot-purified goat antibody to cyclin E1. Panel B,
recombinant cyclin E1 was incubated for 20 min in a CSF extract
supplemented with [-
P]ATP and then
immunoprecipitated. The cyclin E1 immunoprecipitates were blocked (lane a), untreated (lane b), or treated with buffer
alone (lane c), PP1 catalytic subunit (lane d), or
PP1 and 10 µM microcystin (lane e). The samples
were resolved by SDS-polyacrylamide gel electrophoresis and the gel
exposed to film.
To directly investigate the nature of cyclin-cdk complexes in eggs, the various cyclins were immunoprecipitated from a CSF metaphase-arrested extract made from Xenopus eggs and probed for either Cdk2 or Cdc2 and assayed for H1 kinase activity (Fig. 7). Cdk2, but not Cdc2, was detected in cyclin E1 immunoprecipitates; the Cdk2 form with faster mobility, that is thought to be the active form(72, 73) , was particularly evident in the cyclin E1 immunoprecipitates. Cdc2 was identified in immunoprecipitates of B1 and B2. Neither Cdk2 nor Cdc2 were detected in cyclin A1 immunoprecipitates and only a low level of associated kinase activity was measured (Fig. 7, legend). The modest activity is attributable to the low amount of cyclin A1 present in egg extracts arrested in metaphase of meiosis II (Fig. 8A, see also Fig. 9A); cyclin A1-associated kinase activity becomes significantly greater after an extract is released from M phase arrest(30) . In Cdk2 purified from a CSF metaphase-arrested extract (18) , cyclin E1 but not cyclin A1 was detected by immunoblotting (data not shown). While these immunoprecipitations completely depleted the respective cyclins from the extract, the kinase subunits were not appreciably depleted and were readily detected in the supernatants (Fig. 7). The Cdk2 and Cdc2 unassociated with cyclins probably represent the inactive, monomeric cdk pools present in egg extracts (47, 78) .
Figure 8:
Cyclin
E1 and cyclin A1 complexes in oscillating extracts. Panel A,
total histone H1 kinase activity in a CSF extract after the addition of
Ca; asterisks mark time points further
analyzed in panels B and C. Below the graph is the
same extract immunoblotted for cyclin A1. Panel B, cyclin E1
and cyclin A1 immunoprecipitates immunoblotted for the presence of Cdk2
and Cdc2. Panel C, Cdc2 immunoprecipitates immunoblotted for
the presence of cyclin E1 and cyclin A1. Panel D, from a
second CSF extract time course, cyclin E1 was immunoprecipitated at the
indicated times and the precipitates assayed for Cdk2 and associated H1
kinase activity. In the immunoblot two forms of Cdk2 were
resolved.
Figure 9: Characterization of cyclin E1 and cyclin A1 during Xenopus embryogenesis. Panel A, immunoblots of eggs and staged embryos were probed with antibodies to cyclin E1 and cyclin A1. Panel B, the same samples as in panel A were immunoprecipitated with goat preimmune serum, affinity-purified antiserum to cyclin E1, or affinity-purified antiserum to cyclin A1. Shown is the precipitated fraction that was then immunoblotted for Cdk2.
This analysis was extended through a CSF extract time course. The pattern of total H1 kinase activity in the extract used for these experiments is shown in Fig. 8A: the extract progressed from metaphase with high total H1 kinase activity, into interphase with low H1 kinase activity, and then cycled back into M phase with high H1 kinase activity. Also shown is the pattern of cyclin A destruction and synthesis in the same extract. As in the extract analyzed in Fig. 5C, the overall level of cyclin E1 did not change during this extract time course (data not shown). Cdk2 was detected in cyclin E1 immunoprecipitates from all the time points tested (Fig. 8B). As shown in Fig. 8D, the association of cyclin E1 with Cdk2 did not vary, even when the associated kinase activity did oscillate. The mobility of the associated Cdk2 did vary: the faster migrating, more active, species of Cdk2 was not very evident at 45 min when there was the least cyclin E1-associated kinase activity. In human cells, cyclin E-associated kinase activity also correlates with co-precipitation of the most rapidly migrating form of Cdk2(20) . Cdc2 was detected in cyclin A1 immunoprecipitates as A1 became more abundant (Fig. 8B). In keeping with these findings, cyclin A1, but not cyclin E1, was identified in Cdc2 immunoprecipitates (Fig. 8C).
The main finding of this study is that cyclin E forms an
active kinase complex with Cdk2 during meiotic and early embryonic cell
cycles in Xenopus. Originally, cyclin E was cloned by its
complementation of yeast mutants defective in the G/S
transition(2, 3) . In HeLa and B cell lines, the
transcript abundance, protein level, and associated kinase activity of
cyclin E are maximal in late G
and early
S(20, 22, 23, 25) . Meiotic and
embryonic cell cycles, however, lack a G
phase and
therefore cyclin E may have cell cycle functions besides control of the
G
/S transition.
The predicted amino acid sequence of the Xenopus cyclin E1 gene product is 60% identical to the human
cyclin E protein. Homology extends throughout the proteins, and the
cyclin boxes of frog and human cyclin E are virtually identical (Fig. 2A). Northern analysis revealed that the mRNA for cyclin E1 is present throughout early embryogenesis and shows
no apparent transcriptional regulation (Fig. 3). This mRNA
species is a maternal transcript since global activation of zygotic
transcription does not occur until the midblastula transition at stage
8(81) . The mRNA identified by the cyclin E1 probe
declines by stage 13 (data not shown). Presumably zygotically-encoded
forms of cyclin E emerge. This regulation of cyclin E1 gene expression is comparable to that seen during the earliest
stages of Drosophila development. In Drosophila, the
maternally-supplied cyclin E transcripts (type II) are present
constitutively during the early cleavage divisions and degraded during
the cellular blastoderm stage(65) . The zygotic cyclin E mRNA (type I) then appears and is regulated at the transcriptional
level: the transcripts are constitutively present in cell types that
lack a G phase; the transcripts are cell-cycle regulated,
peaking just before S phase in neural cells which have a G
phase; expression is absent in nonproliferating
cells(65) .
During Xenopus oocyte meiotic maturation cyclin E1 protein increased at least 5-fold (Fig. 4B). Cyclin E1 showed a different pattern of accumulation than the mitotic cyclins in cycling egg extracts: A1, B1, and B2 cyclins increase during interphase and are degraded at the conclusion of mitosis(8, 30) , whereas cyclin E1 appeared to be stable through the cell cycle transitions (Fig. 5C). There was a gradual increase in the level of cyclin E1 protein during the first few cleavages (Fig. 9A and data not shown). The protein persisted to the midblastula transition (stage 8), but was undetectable after gastrulation commenced (stage 10.5).
Multiple forms of cyclin E1 emerged during oocyte
maturation (Fig. 4B) and were evident during extract
time courses (Fig. 5C). In human cells antiserum to
human E also identifies a broad band of polypeptides migrating with
apparent molecular masses of 48-51 kDa(23) . Differential
phosphorylation of cyclin E occurs in mammalian
systems(20, 22) . Analogously, the various cyclin E1
species could represent different phosphorylation states. Cyclin E1 was
immunoprecipitated from a CSF extract at time 0 when cyclin E1 is the
most retarded in gels and treated with protein phosphatases (Fig. 6A). There was a downward shift of cyclin E1 that
was completely prevented by the addition of the phosphatase inhibitor
microcystin to the assays. During incubation in CSF extracts,
recombinant cyclin E1 could be labeled with
[-
P]ATP and its apparent molecular weight
in gels significantly increased (Fig. 6B and data not
shown). PP1 selectively removed radiolabel from the most shifted form (Fig. 6B). During extract time courses, the presence of
slower mobility forms coincided with higher, rather than lower,
associated H1 kinase activity in Xenopus cyclin E1
immunocomplexes. The electrophoretic mobility of cyclin E1 appeared to
directly reflect the activity of the complex.
Several criteria suggest that cyclin E1 is the partner of Cdk2 during meiosis and early embryonic cycles. During meiotic maturation the activity of cyclin E1-associated kinase reached a significant level only at meiosis II (Fig. 4D). Previous data from our laboratory showed that Cdk2 kinase activity is not markedly elevated until entry into meiosis II(47) . In a cycling extract, the pattern of cyclin E1-associated kinase activity (Fig. 5A) was similar to that seen for Cdk2(74) . Cdk2 was detected in cyclin E1 immunoprecipitates from egg extracts and cleaving embryos (Fig. 7Fig. 8Fig. 9). Finally, cyclin E1, but not cyclin A1, was detected by immunoblotting of a kinase-active Cdk2 complex purified from Xenopus eggs (data not shown). Cyclin E1 was never identified with Cdc2: cyclin E1 and Cdc2 did not co-immunoprecipitate ( Fig. 7and Fig. 8) and depletion of cyclin E1 from an extract had no effect on Cdc2 kinase activity (data not shown). Instead, the cyclin partners for Cdc2 were A1, B1, and B2 ( Fig. 7and Fig. 8).
It is interesting that cyclin A, a
Cdk2 partner in somatic cell cycles (21, 25, 26) , was not found to associate
with Cdk2 in Xenopus eggs, cycling extracts, or during early
embryogenesis. The reason for lack of association in eggs is not simply
because cyclin A1 exists at extremely low levels, as suggested by
others(71, 78) . Using a high-titer antibody we found
that a maternal store of cyclin A1 was detectable even in the resting
stage VI oocyte, and increased after the completion of meiosis I to a
final concentration of 18 nM in the egg. Data revealed that
cyclin A1 was also complexed to an active histone H1 kinase during
meiotic maturation, with activity being greatest at metaphase arrest of
meiosis II; the activity was almost certainly that of the cyclin A-Cdc2
complex. Cyclin A1 and its associated kinase activity have been found
to increase substantially, however, in extracts released from metaphase
II arrest (8, 30) and in embryos after
fertilization. The associated kinase subunit for cyclin A1
continued to be Cdc2 rather than Cdk2 (Fig. 8). It is unclear
why cyclin A associates with Cdk2 during somatic cycles but not during
meiosis and early embryonic mitoses. One possibility is that in the
maturing oocyte, the severalfold more abundant cyclin E1 competes with
cyclin A1 and establishes a meiotic function for Cdk2, whereas in
somatic cells these two cyclins accumulate at different times in the
cell cycle. However, even when cyclin A1 and E1 are of approximately
equal abundance, as during interphase in a CSF extract, A1 is partnered
with Cdc2 and E1 with Cdk2 (Fig. 8, B and C).
While complexes of recombinant cyclin A1 and Cdk2 can be
made(78) , recombinant cyclin A1 preferentially binds to Cdc2
rather than to Cdk2 when combined in an egg extract(71) .
Another explanation is that there are different cyclin A gene products
present during the early stages of development and in somatic cells
that have different relative affinities for Cdk2 and Cdc2(71) . (
)
What is responsible for the changes in associated
kinase activity of Cdk2-cyclin E1 complexes in oocyte and egg extract
time courses? During oocyte maturation, synthesis of both proteins may
be the explanation for the increase in kinase activity; a necessity for
activating phosphorylation or inactivation of inhibitors may also play
roles because in stage VI oocytes both proteins were detectable but
activity was found to be negligible (Fig. 4). In extracts,
cyclin E1 and Cdk2 protein levels were stable, yet kinase activity did
oscillate (Fig. 5A, and data not shown). Extracts
treated with protein synthesis inhibitors such as emetine (Fig. 5B) and cycloheximide (data not shown) did not
show the reactivation of cyclin E1 complexes seen in control extracts,
even though cyclin E1 protein was quite stable under these conditions (Fig. 5C). Cyclin E1-associated kinase activity was
much reduced in extracts treated with the DNA polymerase inhibitor
aphidicolin (Fig. 5B), suggesting that cyclin
E1-associated kinase activity may be sensitive to DNA checkpoint
control. Post-translational modification of Cdk2 on tyrosine residues (74) or on threonine 160 (72, 73) may be
important in the regulation of Cdk2-cyclin E1 complex activity. In
fact, in the interphase lower activity complex, the fraction of Cdk2 in
the slower migrating, less active form was greater than in M phase (Fig. 7D). The activity of inhibitory proteins such as
p21 and p27 may also be involved in regulation of
complex activity(82, 83) . Homologues of the low
molecular weight cdk inhibitors have recently been identified in Xenopus eggs. (
)
CSF is the activity that
maintains vertebrate eggs arrested at metaphase of MII with high MPF
activity. The product of the Xenopus c-mos proto-oncogene, a serine/threonine kinase, is required but not
sufficient for CSF activity(15, 16, 17) . The
presence of CSF activity at metaphase of meiosis II correlates with
activation of Cdk2-cyclin E1 complexes (Fig. 4D).
Previous studies have shown that ablation of Cdk2 expression with
antisense oligodeoxynucleotides does not affect entry into meiosis I or
II but does cause failure to arrest at metaphase of meiosis II;
metaphase arrest can be restored by addition of purified
Cdk2(18) . Potentially, CSF arrest could be the consequence of
the unique presence at metaphase II of both c-Mos and Cdk2
activities(18) . Preliminary data indicate that the addition of
recombinant cyclin E1 with a subsequent increase in cyclin
E1-associated kinase activity modestly slows the decline in cyclin
B1/B2-associated kinase activity seen after calcium treatment of
CSF-arrested extracts. Cyclin E1 thus appears to stabilize CSF
activity. As cyclin E1 does not bear a destruction box, it is unlikely
that the additional cyclin E1 protein in these extracts is simply
competing for mitotic cyclin degradation directly. Consistent with
these functions, when cyclin E is ectopically expressed in Drosophila, there is an additional cell cycle that is marked
by stabilization of the mitotic cyclins(84) . Such
stabilization may reflect a role for cyclin E complexes in limiting the
period of cyclin instability in Drosophila embryos analogous
to the stabilization of B-type cyclins during CSF arrest in Xenopus.
The cyclin E1-Cdk2 complexes formed in meiosis may
also function after fertilization to support DNA replication in Xenopus embryos. DNA replication potential appears after GVBD
and depends on new protein synthesis(85, 86) ; this
timing coincides with the appearance of active Cdk2-cyclin E1
complexes. After Xenopus egg extracts are depleted of Cdk2
complexes, DNA replication is blocked(51, 76) . If
complexes of Cdk2 with cyclin E1 or cyclin A1, but not cyclin B1, are
then added to such extracts, DNA synthesis is restored. Although there is evidence for a role for cyclin A in DNA
synthesis in tissue culture cells(28, 29) , cyclin A1
appears to be unnecessary for DNA synthesis in Xenopus egg
extracts(51) . As the predominant partner of Cdk2 during early
embryogenesis, cyclin E1 is more likely than cyclin A1 to play a role
in DNA synthesis. Recent genetic studies in Drosophila indicate that cyclin E, rather than cyclin A(87) , plays
an essential role in S phase: cyclin E-deleted Drosophila embryos fail to progress through S phase once maternal stores are
depleted(84) .
This work demonstrates that in Xenopus, Cdk2-cyclin E1 complexes are active during meiotic
and embryonic cycles, both of which lack G phases. In egg
extracts and through the early embryonic cleavage stages, Cdk2 and
cyclin E1 proteins are present constitutively (Fig. 5C,
8B, and 9). Furthermore, the associated kinase activity is
always substantial and exhibits only a modest 2-3-fold
oscillation in activity (Fig. 5A). During Drosophila development, when cyclin E transcripts are
constitutively present, G
phases are undetectable and when cyclin E gene expression is periodic, G
phases are
present(65) . Experiments in which cyclin E gene
expression has been manipulated in Drosophila suggest that it
is the down-regulation of cyclin E that allows exit from rapid
embryonic mitotic cycles and entry of cells into an extended G
phase(85) . Constitutive overexpression of cyclin E in
mammalian cells shortens the duration of G
by 25-33%
and causes premature entry into S phase(33, 34) . This
suggests the possibility that the constitutive activity of cyclin
E-Cdk2 in early embryos may account for the lack of a detectable
G
phase. Cyclin E1 was found to be down-regulated as Xenopus embryos passed the midblastula transition (Fig. 9A). From this point inhibitory tyrosine
phosphorylation of Cdc2 reappears
after being absent during
the rapid cleavage divisions(88) , and a small but growing
proportion of cells are in the G
phase (52) . This
appearance of a G
phase may well require the more regulated
pattern of cyclin E that ensues following activation of zygotic
transcription.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L23857[GenBank].