©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Maternal Xenopus Cdk2-Cyclin E Complexes Function during Meiotic and Early Embryonic Cell Cycles That Lack a G Phase (*)

(Received for publication, August 16, 1994; and in revised form, January 13, 1995)

Rachel E. Rempel (§) Susan B. Sleight James L. Maller (¶)

From the Howard Hughes Medical Institute and Department of Pharmacology, University of Colorado School of Medicine, Denver, Colorado 80262

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(1) in somatic cells.


INTRODUCTION

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), (^1)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(2)/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.(^2)(^3)Another Cdk2 cyclin partner is cyclin E. In tissue culture cells, Cdk2-cyclin E complexes assemble in the middle of G(1), with associated kinase activity peaking in late G(1) and extending into early S phase(20, 22) . Overexpression of human cyclin E in mammalian fibroblasts shortens the duration of the G(1) period, suggesting that cyclin E may be rate-limiting for G(1) progression(33, 34) . Additionally, inhibition of Cdk2-cyclin E complex activity by transforming growth factor-beta in mammalian cells blocks G(1) progression(35) . A G(1) 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(1) events that allow G(1) 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(1)-phase cells(52) .


EXPERIMENTAL PROCEDURES

Preparation of Oocytes, Egg Extracts, and Embryos

Stage VI oocytes were manually dissected from their follicular envelopes and induced to mature by addition of progesterone to 10 µM. Oocytes were removed at the indicated times, homogenized in 10 volumes of extraction buffer (EB), centrifuged for 5 min in a microcentrifuge, and the supernatants stored at -80 °C until further analysis. EB is composed of 80 mM beta-glycerophosphate, 20 mM Hepes, pH 7.5, 15 mM MgCl(2), 20 mM EGTA, 1 mM dithiothreitol, 1 mM Pefabloc (Boehringer Mannheim), 3 µg/ml leupeptin, 50 mM NaF, 1 mM sodium vanadate, 0.2 mM ammonium molybdate, and 30 mMp-nitrophenol phosphate.

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(2) 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 times MMR (10 mM NaCl, 0.2 mM KCl, 0.1 mM MgSO(4), 0.2 mM CaCl(2), and 0.5 mM Hepes, pH 7.8), and then cultured at room temperature in 0.1 times 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.

Cloning of Xenopus Cyclin E

A human cyclin E cDNA(3) , kindly provided by Dr. S. Reed (The Scripps Research Institute, La Jolla, CA), was used as a probe to identify a Xenopus cyclin E gene. The probe extended from BamHI to SacI within the translated region of the human cDNA and was radiolabeled using a random hexamer kit (Boehringer Mannheim). A Xenopus oocyte cDNA library in the gt10 vector(55) , kindly provided by Dr. D. Melton (Harvard University, Cambridge, MA), was then screened. Hybridization and washing steps were essentially as described in the Stratagene protocol manual for Lambda Zap vectors (Stratagene, La Jolla, CA), except that conditions of reduced stringency were used. The filters were hybridized overnight at 42 °C in a buffer consisting of 20 mM Pipes, pH 6.5, 800 mM NaCl, 20% formamide, 0.5% SDS, and 0.1 mg/ml denatured salmon sperm DNA. The nitrocellulose filters were washed five times in 6 times SSC (900 mM NaCl plus 90 mM sodium citrate, pH 7.0) and 0.5% SDS at 50 °C. Several positive clones were twice plaque-purified. The cDNA inserts were excised with EcoRI and subcloned into pUC19 for DNA sequencing. The cDNA clone that demonstrated the greatest homology to the human cyclin E gene was further characterized. This clone gave rise to two EcoRI fragments: the 1497-base pair EcoRI fragment was completely sequenced on both strands and proved to contain the entire coding region for cyclin E; the other EcoRI fragment of approximately 1 kilobase pairs was partially sequenced and presumably encodes 3`-untranslated DNA.

Developmental Northern Analysis

Total RNA was isolated from oocytes, unfertilized eggs, and staged embryos. The RNA was prepared by homogenizing frozen samples in guanidinium isothiocyanate, extracting with acidic phenol, chloroform, and isoamyl alcohol, and precipitating in isopropyl alcohol(56) . The RNA (10 µg/lane) was fractionated by electrophoresis through a horizontal 1.2% agarose-formaldehyde gel and blotted under standard conditions(57) . To determine the migration of the molecular weight markers, the portion of the gel with the RNA molecular weight markers (Life Technologies, Inc.) was stained in ethidium bromide (1 µg/ml) and illuminated by shortwave UV light. The DNA probe was the 1497-base pair (coding) fragment of the cyclin E1 cDNA radiolabeled using a random hexamer kit (Boehringer Mannheim). Hybridization and wash conditions were of high stringency. The blot was then stripped of the cyclin E1 probe (57) and probed with c-src to control for RNA loading(58) . This probe was the 1300-base pair EcoRI fragment of the Xenopus src-1 cDNA(59) , excised from the cDNA clone kindly provided by Dr. R. Steele (University of California, Irvine, CA).

Preparation of Recombinant Cyclin E1

The Xenopus cyclin E1 gene was cloned into a bacterial expression vector that provides a N-terminal six-histidine residue tag. A NheI site was engineered by polymerase chain reaction-directed mutagenesis just upstream of the initiator methionine and this alteration verified by sequence analysis. The cyclin E1 gene fragment extending from NheI to HindIII, a unique site just 3` of the stop codon, was cloned into the pRSET C expression vector (Invitrogen, San Diego CA). Tagged recombinant cyclin E1 protein is larger by 15 N-terminal amino acids than endogenous cyclin E1. BL21(DE3) bacteria were transformed with the cyclin E1 expression plasmid. Cultures were incubated at 37 °C until the A reached 0.5, shifted to room temperature, and supplemented with 0.1 mM isopropylthiogalactopyranoside for 4 h to induce expression of cyclin E1. Bacteria were lysed in a buffer of 50 mM Tris, pH 8.0, 100 mM KCl, 10 mM MgCl(2), 5 mM beta-mercaptoethanol, 0.01% Brij, 1 mg/ml lysozyme, and 10 µg/ml each leupeptin, pepstatin, and chymostatin. After extensive sonication and treatment with DNase I (5 µg/ml) and additional MgCl(2) (10 mM), insoluble protein was pelleted by centrifugation at 10,000 times g for 20 min. Soluble protein was batch loaded onto Ni-NTA-agarose (Quiagen, Chatsworth CA) and mixed at 4 °C for 2 h. The beads were washed with lysis buffer alone, lysis buffer plus 20 mM imidazole, pH 7.0, and lysis buffer plus 40 mM imidazole. Cyclin E1 was eluted from the beads by lysis buffer plus 250 mM imidazole. This fraction was dialyzed against 50 mM Tris, pH 7.6, 100 mM KCl, 1 mM DTT, and 0.01% Brij. The protein was concentrated to 100-400 ng/µl using a Centricon 30 concentrator (Amicon, Beverly MA) and stored in aliquots at -80 °C. Insoluble cyclin E1 was isolated and solubilized as described previously for cyclin B2(60) , except that the final dialysis of cyclin E1 protein was against 20 mM Tris-HCl, pH 7.4, 50 mM NaCl. This solubilized preparation of cyclin E1 was used as antigen for antibody generation and purification, and as a blocking protein.

Antibody Preparation and Immunodetection

Cyclin E1 antiserum was raised both in a goat and in a rabbit using full-length recombinant Xenopus cyclin E1 as the antigen. For immunoprecipitation of cyclin E1, goat immune serum was affinity-purified on a column of Sepharose 4B (Pharmacia Biotech Inc., Piscataway NJ) coupled to full-length recombinant cyclin E1. For immunodetection of cyclin E1, immune goat or rabbit serum was affinity purified on nitrocellulose blots of full-length recombinant cyclin E1 as described below(61) . Insoluble recombinant cyclin E1 was fractionated on 10% SDS-polyacrylamide gels (62) and transferred to nitrocellulose. Blots were stained with 0.2% Ponceau red in 3% trichloroacetic acid and the band of cyclin E1 excised. The cyclin E1 strips were neutralized in 1 times PBS (154 mM NaCl, 7.2 mM Na(2)HPO(4), pH 7.2), blocked in blotto (10% non-fat dry milk in PBS) for 2 h, and incubated with 50% serum in PBS overnight at 4 °C. The strips were washed with PBS, 0.05% Tween 20 and the protein-bound antiserum eluted by incubating the blot with 0.1 M glycine, pH 2.5, for three 10-min periods. The eluates were neutralized by the addition of one-tenth volume of 1 M Tris-HCl, pH 8.0, treated with one-tenth volume of 10 times PBS, supplemented with bovine serum albumin to a final concentration of 1%, and filter sterilized. The blot-purified antiserum was diluted in blotto and could be reused several times.

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 times 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(6)-reactive antibodies was used to allow direct comparison of the signal generated by His(6)-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(6)-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).

Assay of Cyclin E1-associated Kinase Activity and Total Histone H1 Kinase Activity

Immunoprecipitation of cyclin E1-associated kinase activity was performed essentially as described for the assessment of Cdk2 activity(47) . In brief, CSF extract (5-10 µl) or oocyte extract (derived from 10 oocytes) was diluted in EB to a final volume of 150 µl, precleared with protein G-Sepharose (Sigma), incubated with 1 µl of affinity-purified goat antibody for 2 h on ice, mixed with 25 µl of 50% protein G-Sepharose for 1 h, washed with low and high salt buffers (20 mM Tris-HCl, pH 7.4, 5 mM EDTA, 0.1% Triton X-100, and either 100 mM or 1 M NaCl, respectively), and finally washed with kinase assay buffer (20 mM Hepes, pH 7.5, 15 mM MgCl(2), 5 mM EGTA, 1 mM DTT). At various time points, samples were immunoprecipitated with affinity-purified antibody blocked by preincubation with recombinant cyclin E1 (10 µg/µl serum) to obtain a background level of nonspecific kinase activity. Isolation of cyclin A1- and cyclin B1/B2-associated kinase activities relied on the use of the appropriate sheep immune sera; for determination of Cdc2 activity, rabbit immune serum was used and the complexes isolated with protein A-Sepharose.

Immunoprecipitates were incubated with 25 µl of a buffer containing 20 mM Hepes, pH 7.5, 15 mM MgCl(2), 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 times sample buffer(62) . For assays of total histone H1 kinase activity, 3 µl of extract was diluted 10-fold in buffer (80 mM beta-glycerophosphate, 15 mM MgCl(2), 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(2), 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 times 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.

Western Analysis of Immunoprecipitates

Samples from a CSF extract time course (10 µl) or staged embryos (10 embryos equivalent) were diluted in EB supplemented with 1 µM microcystin and 10 mg/ml bovine serum albumin or ovalbumin. As described above, kinase-active complexes were isolated from the extracts using affinity-purified goat antibody to cyclin E1, affinity-purified sheep antibody to cyclin A1, sheep immune serum to cyclin B1 plus sheep immune serum to cyclin B2, or rabbit immune serum to Cdc2. The supernatant and precipitated fractions were treated with sample buffer, boiled, fractionated by electrophoresis, transferred to nitrocellulose, and probed with the designated antibodies. In some instances duplicate precipitated fractions were assayed for associated histone H1 kinase activity.

Phosphatase Treatment of Cyclin E1 Immunoprecipitates

Endogenous cyclin E1 was immunoprecipitated from a CSF extract arrested in metaphase of meiosis II using rabbit immune serum. These immunoprecipitates were washed as for kinase assays using buffers supplemented with 50 mM NaF, 30 mMp-nitrophenyl phosphate, and 0.5 mM phenylmethylsulfonyl fluoride. For radiolabeling of cyclin E1, recombinant soluble cyclin E1 (1 µg) and [-P]ATP (1 mCi/ml) were added to a CSF-arrested extract (40 µl) and incubated for 40 min. The extract was treated essentially as described previously in Erikson and Maller (64) except that in buffer A sodium deoxycholate was omitted. Radiolabeled cyclin E1 was recovered using affinity-purified goat immune serum and protein G-Sepharose; some sample was treated with antibody blocked by prior incubation with recombinant cyclin E1. The washes were as described (64) followed by an additional wash with a buffer containing 20 mM Tris-HCl, pH 7.4, 1 mM EDTA, and 200 µM Pefabloc.

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% beta-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.


RESULTS

Cloning of Xenopus Cyclin E and Expression during Early Xenopus Development

A human cyclin E cDNA was used to identify the Xenopus cyclin E homolog by low stringency hybridization screening. The 2.5-kilobase pair cDNA clone isolated has a complete open reading frame of 1224 bases that predicts a 408-amino acid protein with a calculated M(r) of 47,168. The nucleotide sequence for Xenopus cyclin E1 and the predicted amino acid sequence are shown in Fig. 1. This clone is designated E1 because there are presumably additional cyclin E genes expressed at later developmental stages. The predicted Xenopus cyclin E1 gene product shows strong homology with the human cyclin E gene product. They are 60% identical at the amino acid level, with similarity extending throughout the proteins; the identity rises to 85% when the cyclin box domains are compared (Fig. 2A). The Drosophila type I cyclin E and the Xenopus protein show a lesser degree of homology, having 68% identity when the cyclin box domains are compared(65) . Both Drosophila cyclin E proteins, the type I zygotic form, and the type II form derived from maternal transcripts, are significantly larger than Xenopus E1, with 601 amino acids and 708 amino acids, respectively(65) . Homology of Xenopus cyclin E1 to the Xenopus mitotic cyclins A1, B1, and B2 is largely restricted to the cyclin box domain (Fig. 2B), and there is less than 19% overall identity between cyclin E1 and any of the other cyclins. Cyclin E1 does not bear the destruction box (66) found in the mitotic cyclins and also does not feature other hallmarks of proteins with short half-lives, such as PEST sequences(67) . In contrast, the human and Drosophila E cyclins do bear PEST sequences and are expected to be rapidly degraded(65, 68) .


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(1)/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.



Antisera Specific for Xenopus Cyclin E1

Previous work established that the endogenous cyclin partner for Cdk2 in oocytes and egg extracts is not cyclin A1, B1, or B2(8, 47, 51) . Even when egg extracts are supplemented with exogenous A- and B-type cyclins most if not all kinase activity that associates with them is due to Cdc2 (69, 70, 71) . Cyclin E was deemed the most promising partner for Cdk2 based on reports in other systems of Cdk2-cyclin E complexes. To characterize the endogenous cyclin E1 protein, polyclonal antibodies against recombinant full-length cyclin E1 were produced both in a goat and rabbit and then affinity purified. These antibodies did not cross-react with other cyclins by immunoblot analysis and did not immunoprecipitate other cyclins from [S]methioninelabeled egg extracts (data not shown). Blot-purified cyclin E1 antiserum was capable of detecting 0.1 ng of recombinant cyclin E1. Detection of antigen, whether endogenous or recombinant cyclin E1, was effectively blocked by preincubation of the blot-purified antibody with recombinant cyclin E1 protein (Fig. 4A).


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.



Synthesis of Cyclin E1 and Kinase Activation during Progesterone-induced Oocyte Maturation

If cyclin E1 is the meiotic partner of Cdk2, cyclin E1 protein should be present when Cdk2 complexes are active. To evaluate this prediction, the levels of Cdk2 and cyclin E1 proteins during oocyte maturation were determined by immunoblot analysis. Increased synthesis of Cdk2 during maturation has been reported previously (47) but not in relation to the accumulation of cyclins. As shown in Fig. 4C, Cdk2 was present at a low level as a single molecular weight band in resting oocytes (stage VI). Following progesterone addition, the level of Cdk2 increased approximately 5-fold. In other blots, a faster migrating form of Cdk2 was resolved as the oocytes progressed into meiosis II (data not shown) and in egg samples (Fig. 7A). The lower band of Cdk2 has been proposed to be the kinase-active form, phosphorylated on threonine 160(72, 73) . Duplicate samples were immunoblotted for cyclin E1 (Fig. 4B). In common with Cdk2, the level of cyclin E1 was low but detectable in resting oocytes. Cyclin E1 protein was unchanged through meiosis I. During meiosis II, the amount of cyclin E1 increased substantially and there was an incremental appearance of slower migrating forms.

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) .

Cyclin E1-associated Kinase Activity Oscillates during an Egg Extract Time Course

The behavior of cyclin E1 was then assessed in extracts that progress through several cell cycle transitions. As described in the Introduction, the CSF extract cell cycle encompasses exit from M phase, DNA replication, and re-entry into M phase. Such extracts have been used to investigate the role of mitotic cyclin degradation in exit from M phase, the timing of cyclin-dependent kinase activation, and the dependence of mitosis on completion of DNA replication. Cyclin E1- and Cdc2-associated kinase activities were compared during the time course of a CSF extract released from metaphase of meiosis II arrest upon the addition of Ca. As shown in Fig. 5A, cyclin E1-associated kinase activity did oscillate during the cell cycle: it was high in M phase, at its lowest point during interphase, and rose again as the extracts progressed into M phase. As was found for Cdk2(51, 74) , the activity varied only 2-3-fold rather than the 10-fold seen for Cdc2 and appeared to be reactivated earlier than Cdc2. Inhibition of DNA synthesis by the addition of the DNA polymerase inhibitor aphidicolin and high numbers of nuclei has been found to block the reactivation of Cdk2 (74) as well as Cdc2 kinases(75) . In such extracts cyclin E1-associated kinase activity, like that of Cdk2 activity, was not reactivated (Fig. 5B). Cyclin E1-associated kinase activity was also not reactivated in protein synthesis-inhibited extracts (Fig. 5B).


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. (^4)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.

Cyclin E1 Protein Is Stable and Differentially Phosphorylated during the Cell Cycle

The oscillation in cyclin E1-associated kinase activity could reflect changes in the level of cyclin E1 protein. However, when a CSF extract was treated with Ca to enter the cell cycle and immunoblotted for cyclin E1, no dramatic changes in the level of cyclin E1 protein were evident (Fig. 5C, i). Even when extracts were treated with emetine, a protein synthesis inhibitor, cyclin E1 remained stable, with a decline in protein apparent only at 2 h (Fig. 5C, ii). The level of cyclin E1 was estimated by quantitative immunoblotting as described under ``Experimental Procedures.'' In a CSF metaphase-arrested extract, cyclin E1 was estimated to be 65 nM; in an emetine-treated extract 40 min after addition of Ca the level of cyclin E1 was relatively unchanged, approximately 47 nM. These estimates for cyclin E1 are substantially higher than those reported for the A1, B1, and B2 cyclins in unfertilized eggs (78, 79) . The level of cyclin A1 was also determined using high affinity cyclin A1 antibodies: cyclin A1 was estimated to be 18 nM in a CSF metaphase-arrested extract, was undetectable within 10 min of release from CSF arrest, and rose to 60 nM within 45 min of release from CSF arrest.

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.



Cyclin E1 Is Complexed with Cdk2

Both A- and E-type cyclins have been reported to associate with Cdk2 in a variety of species(2, 19, 20, 21, 22, 23, 33, 35, 51) . However, the parallel patterns of associated kinase activity seen for cyclin E1 and Cdk2 immunoprecipitates during oocyte maturation and in egg extracts suggested an association of cyclin E1 rather than cyclin A1 with Cdk2. Furthermore, it was reported previously that in egg extracts newly synthesized A1, B1, and B2 cyclins are not associated with Cdk2, but rather with Cdc2(8, 47, 51) .

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).

Cdk2-Cyclin E1 Complexes Persist Through Early Embryogenesis

Analysis of cyclin E1 complexes was extended into the early mitotic cycles of cleaving embryos (Fig. 9). The early cell cycles of embryos are rapid, synchronous, and feature a simple oscillation between S phase and M phase(80) . Cyclin E1, just detectable in oocytes and plentiful in eggs and CSF extracts, was also present in the early embryonic stages (Fig. 9A). Between stage 8 and stage 10.5, cyclin E1 disappeared. Cyclin E1-associated kinase activity also declined after stage 8, approximately at stage 9. (^5)Cyclin A1 remained detectable for somewhat longer during embryogenesis (Fig. 9A). As in extracts, cyclin E1 rather than A1 was the predominant partner for Cdk2 (Fig. 9B). In the most advanced stage analyzed (St 8) there was some portion of Cdk2, just above background, associated with cyclin A1.


DISCUSSION

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(1)/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(1) and early S(20, 22, 23, 25) . Meiotic and embryonic cell cycles, however, lack a G(1) phase and therefore cyclin E may have cell cycle functions besides control of the G(1)/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(1) phase; the transcripts are cell-cycle regulated, peaking just before S phase in neural cells which have a G(1) 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.^5 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) . (^6)

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. (^7)

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.^4 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(1) 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(1) phases are undetectable and when cyclin E gene expression is periodic, G(1) 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(1) phase(85) . Constitutive overexpression of cyclin E in mammalian cells shortens the duration of G(1) 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(1) 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^5 after being absent during the rapid cleavage divisions(88) , and a small but growing proportion of cells are in the G(1) phase (52) . This appearance of a G(1) phase may well require the more regulated pattern of cyclin E that ensues following activation of zygotic transcription.


FOOTNOTES

*
This work was supported by Grant GM26743 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L23857[GenBank].

§
Associate of the Howard Hughes Medical Institute.

Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed.

(^1)
The abbreviations used are: MPF, maturation-promoting factor; CSF, cytostatic factor; EB, extraction buffer; Pipes, 1,4-piperazinediethanesulfonic acid; DTT, dithiothreitol; PBS, phosphate-buffered saline: GVBD, germinal vesicle breakdown.

(^2)
Dr. T. Kishimoto, Tokyo Institute of Technology, Tokyo, personal communication.

(^3)
Dr. M. J. Cockerill and Dr. T. Hunt, Imperial Cancer Research Fund, South Mimms, United Kingdom, personal communication.

(^4)
Dr. J. Blow, Imperial Cancer Research Fund, South Mimms, United Kingdom, personal communication.

(^5)
R. Hartley, R. Rempel, and J. Maller, manuscript in preparation.

(^6)
Dr. T. Hunt, Imperial Cancer Research Fund, South Mimms, United Kingdom, personal communication.

(^7)
J. Su, E. Erikson, and J. Maller, unpublished results.


ACKNOWLEDGEMENTS

We thank Brad Lattes and Andrea Lewellyn for technical assistance, Olivier Haccard for help with computerized sequence analysis, Rebecca Hartley for assistance with embryo staging, and Eleanor Erikson for a critical reading of the manuscript.


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