DEVELOPMENTAL

BIOLOGY

144,54-64 (1991)

A Novel M Phase-Specific HI Kinase Recognized by the Mitosis-Specific Monoclonal Antibody MPM-2 JIANKUANG,* JOSEPHE.PENKALA,-~ DAVID A. WRIGHT,~ GRADY F. SAUNDERS,I$ANDPOTUN.RAO**~ Departments of *Medical Oncology, tMolecular Genetics, and $Biochemistry and Molecular Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas ~030 Accepted November 8, 1990 At the onset of mitosis, eukaryotic cells display an abrupt increase in a Ca’+- and cyclic nucleotide-independent histone Hl kinase activity, referred to as growth-associated or M phase-specific Hl kinase. The molecular basis for this activity is generally attributed to a kinase complex that consists of the ~34”” protein and cyclin, and exhibits maturation-promoting factor (MPF) activity. In the present study, we show that more than one kinase contributes to M phase-specific Hl kinase activity. When matureXenopus oocyte extract prepared with ATPyS and NaF was fractionated by gel filtration, two prominent peaks of Hl kinase activity were detected, with apparent molecular masses of 600 and 150 kDa. The 150-kDa kinase copurified with the ~34~’ protein and was immobilized by the sue 1 gene product p13 and anti-cyclin B2, which are specific for the C&Z kinase complex. However, the BOO-kDakinase did not satisfy any of these criteria, thus identifying it as a novel M phase-specific Hl kinase. Only the 600-kDa kinase was recognized by the mitosis-specific monoclonal antibody, MPM-2, which inhibits Xenopus oocyte maturation and immunodepletes MPF activity. Furthermore, not only did the full activation of this kinase (MPM-2 kinase) coincide with the activation of MPF during the cell cycle, but also MPM-2 kinase-positive fractions obtained by gel filtration accelerated progesterone-induced oocyte maturation. It is, therefore, likely that MPM-2 kinase is a positive regulator in the M phase induction pathway. 0 1991 Academic Press, Inc.

The initiation of M phase also correlates with the activity of maturation-promoting factor (MPF), which is The eukaryotic cell cycle consists of two morphologihypothesized to be responsible for the interphase to M cally distinct phases: interphase and M phase (mitosis phase transition in all eukaryotic cells (Wasserman and and meiosis). The initiation of M phase always coincides Smith, 1978; Gerhart et uL, 1984). MPF is assayed by its with a high level of protein phosphorylation (Capony et ability to induce immature oocytes arrested at the G2/ al, 1986; Karsenti et al, 1987; Lohka et al., 1987). This M boundary of the first meiotic division to resume meioinvariant correlation has led not only to the idea that sis until they arrest at second meiotic metaphase as maprotein phosphorylation plays an essential role in M ture oocytes. Under physiological conditions, oocyte phase induction, but also to the discovery of the growthmaturation is induced by progesterone, which is seassociated or M phase-specific histone Hl kinase, pres- creted by the follicle cells and leads to the activation of ent in many species (Lake and Salzman, 1972; Lake, MPF, a step that is dependent on new protein synthesis. 1973; Bradbury et ah, 1974; Zeilig and Langan, 1980; When microinjected into immature oocytes, MPF, in the Woodford and Pardee, 1986; Labbe et uZ., 1988a). This absence of protein synthesis, is able to activate an entire kinase, which is independent of Ca2+,cyclic nucleotides, pool of latent MPF, which in turn induces maturation. and diacylglycerol, phosphorylates serine and threonine This event is accompanied by a high level of protein residues and strongly prefers histone Hl as an in vitro phosphorylation, which led to the proposal that MPF substrate. The activity of this kinase oscillates during must be a kinase or an activator of a kinase. both mitotic and meiotic cycles in tandem with the initiDuring the past two years, there has been a major ation and maintenance of M phase (Matthews and advance in determining the molecular basis of both M Huebner, 1985; Picard et uL, 1987; Labbe et uL, 1988a). It phase-specific Hl kinase and MPF activity. Success in was suspected that the M phase-specific Hl kinase plays their purification led to the discovery that they share as an essential role in chromosome condensation at the onan essential component the cdd gene product ~34”~~, set of M phase (Bradbury et al., 1973; Marks et uL, 1973). which encodes a highly conserved protein kinase necessary for entry into mitosis (Arion et uL, 1988; Gautier et uZ., 1988; Labbe et uL, 1988b). When Labbe et al. (1989) 1 To whom correspondence and reprint requests should be adsimultaneously examined Hl kinase and MPF activities dressed. INTRODUCTION

0012-1606/91 $3.00 Copyright All rights

@ 1991 by Academic Press. Inc. of reproduction in any form reserved.

54

KUANGETAL.

Novel M Phase-Specific

in starfish oocytes, they were able to show that the cdc2 protein copurified with both activities during each of six successive chromatographic fractionations. These findings led to the general belief that MPF, the cdc2 kinase, and the M phase-specific Hl kinase are identical (Dunphy and Newport, 1988a; Murray, 1988; Lohka, 1989). As a consequence, many laboratories use the Hl kinase assay as a measure of cdc2 kinase and MPF activity. In most cases, cdc2 kinase exists as a protein complex having an apparent molecular weight of 100 to 200 kDa (Draetta and Beach, 1988; Pondaven et ah, 1990). Cyclins, a family of proteins that are synthesized during interphase and degraded at the end of mitosis (Evans et al., 1983; Swenson et al., 1986), were shown to be complexed with ~34~~’ (Draetta et ah, 1989; Meijer et al., 1989; Pondaven et al., 1990). Since the synthesis and degradation of cyclins were demonstrated to be required for the activation and inactivation of cdc2 kinase, respectively (Booher et ah, 1989; Minshull et aZ., 1989;Murray et ah, 1989), it is likely that cyclin is a regulatory element in the cdc2 kinase complex. The sue1 gene product p13 has been shown to be a component of a portion of the cd& kinase complexes in viva (Brizuela et al, 1987; Draetta et ah, 1987). Overexpression of the sue1 gene suppresses cd& mutants in an allele-specific manner (Hayles et al., 1986). In vitro, p13 binds to p34cdc2very efficiently (Arion et al., 1988; Dunphy et al, 1988; Brizuela et al, 1989; Meijer et al, 1989), and its binding does not inhibit the Hl kinase activity of the cdc2 kinase complex (Booher et al, 1989). In Xenms egg extract, excess yeast p13 inhibits the activation of cdc2 kinase and mitotic conversion of the extract (Dunphy et al., 1988; Dunphy and Newport, 1989). These results suggest that p13 might also be involved in the regulation of the activation of cdc2 kinase. Our laboratory has been trying to identify mitotic regulators through the use of mitosis-specific monoclonal antibodies. Both MPF and M phase-specific Hl kinase are mitosis-specific activities. Other proteins whose presence or modification is specific to mitotic cells may also be involved in the regulation of the initiation of mitosis and meiosis. By raising monoclonal antibodies that specifically or preferentially react with mitotic cells compared to interphase cells, those mitosis-specific proteins might be identified and their roles in mitotic induction studied. Using mitotic HeLa cell extract as the immunogen, 13 mitosis-specific monoclonal antibodies were obtained and designated MPM-1 to MPM-13. MPM-1 (IgM) and MPM-2 (IgG), which recognize a common epitope, are the best characterized (Davis et al, 1983; Vandre et ab, 1984,1986; Engle et aZ.,1988; Hiraoka et ah, 1989; Kuang et al., 1989; Rao et al, 1989; Vandre and Borisy, 1989;

55

HI Kinase

Centonze and Borisy, 1990). They react with mitotic cells of all species tested (Davis et al., 1983; Vandre et ah, 1986). Immunofluorescence studies show that the antigens to MPM-1 and MPM-2 are distributed throughout the mitotic cell, in both the soluble and the structural fractions. On immunoblots, MPM-1 and MPM-2 recognize a discrete set of polypeptides which are synthesized during interphase and phosphorylated during the G2/M transition (Davis et ab, 1983). It was demonstrated that this phosphorylation is required for MPM-2 recognition of the MPM-2 epitope common to this group of proteins. Recently, we have shown that MPM-2 inhibits Xeno pus oocyte maturation and immunodepletes MPF activity from both mature oocyte extract and mitotic HeLa cell extract (Kuang et al, 1989). These studies identify MPM-2 as a probe for either MPF itself or a regulator of MPF. Therefore, if, as is generally believed, MPF is identical to cdc2 kinase, we would expect MPM-2 to recognize the mitosis-specific phosphorylation of either a component in the cdc2 kinase complex or a factor that regulates cdc2 kinase activity. In an attempt to explore this possibility in the present study, we found that MPM-2 recognizes a distinct M phase-specific Hl kinase, which might be a positive regulator in the M phase induction pathway. MATERIALS

AND

METHODS

Animals Adult female Xenopus Zaevis (oocyte-positive) were obtained from Nasco (Ft. Atkinson, WI). Stage VI oocytes were obtained by surgical removal of ovarian tissue from animals anesthetized in 1.5% ethyl-m-aminobenzoate (Sigma). Oocytes were manually defolliculated in modified Barth’s solution (MBS) (Gurdon, 1976) with watchmaker forceps, while viewed under a dissecting microscope. For experiments requiring hormonally induced maturation, defolliculated oocytes were exposed to 1 pg/ml of progesterone (Sigma) in MBS. Unfertilized eggs were collected from ovulating females which, 12 hr previously, had been injected into the dorsal lymph sac with 800 IU human chorionic gonadotropin (Sigma). Eggs were promptly dejellied in 2% cysteine in 0.1 MTris (pH 7.9), followed by thorough rinsing in MBS. For cell cycle studies, fertilized eggs were obtained by directly pipetting over the eggs a sperm suspension prepared by macerating a testis (surgically removed from an adult male frog) in 1 ml of 1X MBS. After l-2 min, the eggs were flooded with 0.1X MBS, which marked the time of fertilization. Twenty minutes later, following rotation, the eggs were dejellied in preparation for the time course.

56 Preparation

DEVELOPMENTALBIOLOGY

of Oocyte and Egg Extracts

vOLUME144,1991

ing them with unstained molecular weight standards from BRL and Bio-Rad in 12.5% SDS gels.

Extracts from stage VI immature oocytes, unfertilized eggs (mature oocyte extract: MOE), and fertilized eggs were prepared as follows. Oocytes or eggs were P13-Sepharose Abswption and Immunoabsorption rinsed twice in cold extraction buffer (EB: 80 mM soMPM-2 and RDA-1 (nucleolar-specific monoclonal dium ,&glycerophosphate, 20 mMEGTA, 15 mMMgC1,). antibody) ascites were produced as described (Davis et After excess buffer was removed, the oocytes or eggs al, 1983). Anti-frog cyclin B2 antiserum and the preimwere crushed in an equal volume of EB containing 50 mune serum were kindly provided by the laboratory of mMNaF, 2 mMATPys (Boehringer-Mannheim), 10 mM Tim Hunt. IgGs from ascites or serum were immobilized DTT, and a mixture of protease inhibitors (see Kuang et to Protein-A beads (Bio-Rad) using the MAP II Kit al., 1989). In some experiments, ATPyS and NaF were (Bio-Rad). excluded from the buffer. Following extraction, the hoThe fission yeast sue1 gene product p13 covalently mogenates were centrifuged at 100,OOOg for 1 hr at 4°C linked to Sepharose beads (obtained from the laborato(for gel filtration) or at 10,OOOgfor 20 min (for time ries of Tim Hunt and Marc Kirschner) was prepared as course studies). The material between the pellet and the previously described (Dunphy et aZ., 1988). lipid cap was recovered and stored at -70°C if not used The immunoaffinity beads or p13 beads were washed immediately. with EB and then mixed with MOE or other samples by rotation at 4°C for 3 hr. The beads were pelleted and Gel Filtration of Oocyte or Egg Extracts then washed at least five times with EB containing 0.5% NP-40, 0.5 1M NaCl, 20 mlM NaF, 1 mM DTT, and One milliliter of extract was subjected to gel filtration at 4°C on an Ultrogel AcA34 column (Pharmacia; 0.9 1 mMATP. X 59 cm, approximately 40-ml bed volume), preequilibrated in EB containing 1 mMDTT, 1 mMATP, and 20 mM NaF. One-milliliter fractions were collected at a flow rate of 0.3 ml/min. Assay of Hl Kinase Activity

Hl kinase activity in the solution was assayed by mixing 7.5 11 of sample with 2.5 ~1 of Hl kinase assay reaction mixture at 22°C for 30 min. The reaction mixture contained 100 pg/ml histone Hl (Boehringer-Mannheim), 4 mM CAMP-dependent protein kinase inhibitor peptide (Sigma catalog No P3294), 100 puMATP, and 0.5 &i/p1 [-Y-~~P]ATP(New England Nuclear) in EB. The reaction was stopped by the addition of Laemmli gel sample buffer (Laemmli, 1970). Samples were then electrophoresed on a 12.5% polyacrylamide gel and stained with Coomassie blue to visualize the histone Hl bands. Phosphorylation of histone Hl was revealed by autoradiography. The intensities of the 32P-labeled bands on the exposed X-ray film were quantitated with a densitometer (Joyce Loebl). Western Blot Analysis

Proteins were separated by 12.5% SDS-PAGE, then electrophoretically transferred onto nitrocellulose, and immunostained as described (Kuang et al, 1989). Antiserum against the fission yeast cdc2 protein PSTAIR was a generous gift from the laboratory of Paul Nurse. Prestained molecular weight standards from Sigma were used as references for estimating the molecular weights of the MPM-2-reactive peptides, after calibrat-

Assay for MPF Activity

Samples were assayed for MPF activity by their ability to induce oocyte maturation 2 hr after microinjection (70 nl). The induction of maturation was judged by both germinal vesicle breakdown (GVBD) and white spot formation which were determined after fixation of the oocytes in 5% (wt/vol) trichloroacetic acid and dissection. The maximal-fold dilution of the sample with EB that allowed maturation induction in 50% of the injected oocytes was used to express relative MPF activity. RESULTS

MPM-2 Binds Hl Kinase Activity Xenopus Oocytes

in Extract

of Mature

To determine whether MPM-2 recognizes a component in the cd& kinase complex, we first tested whether MPM-2 affinity beads immobilized any Hl kinase activity from M phase extracts that was Ca2’- and CAMP-independent, as is characteristic of cdc2 kinase. For this purpose, we prepared MOE from unfertilized eggs of Xenopus laevis with an EB composed of 80 mM Na /3glycerophosphate, 20 mM EGTA, 15 mM MgCl,, 10 mM dithiothreitol, 50 mM NaF, and 2 mM ATP+. Extract prepared in this manner is able to retain full MPF activity for at least 1 week when stored at 4°C. The extract was absorbed by excess amounts of MPM-2 or control antibody (RDA-1) affinity beads for 3 hr, which allowed the complete depletion of MPM-2 antigens from the extract as assayed by immunoblot. Following absorption,

KUANG ET AL.

123

4

5

Novel M Phase-Speci$c

Histone HI

6

FIG. 1. MPM-2 binds Hl kinase in mature oocyte extract of Xenqpus laevis. Mature oocyte extract was mixed with equal volumes of protein A beads to which MPM-2 or control IgGs were bound. After being rotated at 4°C for 2 hr, the beads were pelleted and washed. Hl kinase activity of the proteins on the beads (A) and in the supernatants (B) was then assayed. Phosphorylation of histone Hl was revealed by autoradiography following separation of the proteins by SDS-PAGE. Lanes 1 and 4: mouse IgG; lanes 2 and 5: MPM-2; lanes 3 and 6: RDA-1.

the beads were washed thoroughly and assayed for Hl kinase activity. We found that although control antibody affinity beads immobilized little or no Hl kinase activity from MOE, proteins on MPM-2 affinity beads exhibited a high level of this activity (Fig. 1A). Assays of the Hl kinase activity remaining in MOE after immunoabsorption showed that extract depleted by MPM2 had noticeably less Hl kinase activity than extract absorbed by the control antibody affinity beads (Fig. 1B). Quantitation of the Hl kinase activity of the absorbed samples by densitometric scanning of the bands representing phosphorylated histone Hl revealed that the reduction in total Hl kinase activity in MOE absorbed by MPM-2 was approximately 20%. Therefore, we conclude that MPM-2 recognizes a factor or factors that contribute approximately 20% of the M phase-specific Hl kinase activity.

57

HI Kinase

was at least 10 times lower in either of the two molecular weight regions (data not shown), indicating that the 600 and 150 kDa kinases were both M phase-specific. The 600-kDa Kinase Inhibitors

was Stabilized

by Phosphatase

Labbe et al. (1988a,b) have reported the recovery of only one Hl kinase peak during chromatography of MOE. In those studies, MPF-active MOE was prepared with EB that did not include NaF and ATPrS. To test whether these agents might have stabilized the 600-kDa kinase that we recovered from gel filtration, we fractionated MOE prepared with or without NaF and ATPrS and tested the Hl kinase activity of the fractions collected. We found that the inclusion of NaF and ATPyS in MOE was unnecessary for the recovery of the 150-kDa Hl kinase, but was required for reproducible recovery of the 600-kDa Hl kinase (Figs. 3A and 3B). These results indicate that the 600-kDa kinase is stabilized by NaF, a phosphatase inhibitor, and ATPyS, an analog of ATP that produces a thiophosphorylated protein resistant to the action of phosphatases. Therefore, the failure to detect this kinase in the past was probably due to its inactivation by dephosphorylation. MPM-2 Recognizes a 600-kDa Hl Kinase that Is not cd&? Kinase

To determine which peak of Hl kinase activity was recognized by MPM-2, the peak fractions for each kinase were pooled and immunoabsorbed by MPM-2 or control antibody affinity beads, and the Hl kinase activity on the beads was assayed. Figure 4A shows that MPM-2 bound high levels of Hl kinase activity from the 600Gel Filtration of MOE Reveals Two Prominent Peaks of kDa fractions, but very little from the 150-kDa fracM Phase-Speci&c Hl Kinase Activity tions. Assay of the supernatants after absorption We considered two alternatives to explain why MPM- showed that the major portion of Hl kinase activity in 2 immobilized only 20% of the M phase-specific Hl ki- the 600-kDa fractions was depleted by MPM-2 (Fig. 4B). nase activity from MOE. Assuming that cdc2 kinase is Therefore, the 600-kDa kinase activity was mainly due the only M phase-specific Hl kinase in MOE, MPM-2 to an MPM-2 antigen, and we will henceforth refer to it might recognize a proportion of cd&? kinase molecules as MPM-2 kinase. To determine which peak of Hl kinase activity was that was phosphorylated at the MPM-2-reactive site. Alternatively, there might be a second M phase-specific due to cdc2 kinase, we first performed Western blot analysis of the fractions from gel filtration, using the Hl kinase in MOE that MPM-2 recognizes. To distinguish between these two possibilities, we fractionated PSTAIR antibody (Lee and Nurse, 1987) that recognizes MOE by gel filtration on an Ultrogel AcA34 column (op- the highly conserved domain of ~34’~“. We observed timum separation range: 20-350 kDa; exclusion limit: that ~34~~” copurified with the 150-kDa kinase, but was 750 kDa) and assayed Hl kinase activity in the fractions undetectable in the 600-kDa fractions containing MPMcollected. We found that Hl kinase activity occurred as 2 kinase (Fig. 2B). Another feature diagnostic of cdc2 two prominent peaks (Fig. 2A), which varied in relative kinase is that it binds to the yeast sucl gene product ~13. intensities among experiments (cf. Figs. 2A and 3A). Thus the peak fractions for each kinase were pooled and The first peak had an apparent molecular weight of 600 absorbed by pl3-Sepharose, after which both the superkDa, and the second peak 150 kDa. When immature oo- natant and the beads were assayed for Hl kinase activcyte extract was similarly analyzed, Hl kinase activity ity. Strikingly, while pl3-Sepharose immobilized little

58

DEVELOPMENTAL BIOLOGY

12

14

16

16

20

22

6 24

VOLUME 144, 1991

26

66 28

30

32

-

~ra~ti011

-Hidme

No. HI

kDa 200 116 90 76 60 FIG. 2. The 150-kDa Hl kinase copurifies with p34”&, but the 600-kDa Hl kinase peak does not. One milliliter of MOE prepared containing 50 mMNaF and 2 mMATPrS was fractionated on an Ultrogel AcA34 column. Fractions were assayed for Hl kinase activity (A). Arrows indicate the elution positions: (1) bovine thyroglobulin (6’70kDa); (2) immunoglobulin G (150 kDa); (3) chicken ovalbumin (44 kDa); (4) horse myoglobin (17.8). The presence of the cd&protein (B) was determined by Western blot analysis using the antiserum against the highly conserved domain of the cd& protein (PSTAIR). (C) An immunoblot stained by MPM-2, where the two prominent immunoreactive peptides of ‘72and 180 kDa are indicated by arrows.

Hl kinase activity from the 600-kDa fractions, it immobilized almost all from the 150-kDa fractions (Fig. 5). Furthermore, since cyclin B2 has been shown to be a component of the cd& kinase complex (Gautier et aZ., 1990), we also tested which peak of Hl kinase activity could be immobilized by anti-frog cyclin B2 immunoaffinity beads. We found that anti-cyclin B2 affinity beads immobilized a much higher level of Hl kinase activity from the 150-kDa fractions than from the 600-kDa fractions, while no Hl kinase activity was immobilized by control antibody affinity beads (Fig. 6). Densitometric scanning showed that the difference in the intensity of the bands was 15-fold. Therefore, the anti-cyclin antibody mainly recognizes the SO-kDa kinase. Together these results indicate that the 150-kDa kinase satisfies

all the criteria for cd&?kinase, while MPM-2 kinase does not. The finding that anti-cyclin affinity beads immobilized some Hl kinase activity from MPM-2 kinase-positive fractions raised the possibility that cyclin might be a component of a portion of the MPM-2 kinase molecules. To examine this possibility, MOE was immunoabsorbed with anti-cyclin B2 affinity beads and the proteins immobilized on the beads were analyzed for the presence of MPM-e-reactive peptides. We found that although anti-cyclin affinity beads immobilized a high level of Hl kinase activity from MOE (Fig. 7A), they did not immobilize any MPM-2-reactive peptides from MOE under conditions where MPM-2 affinity beads immobi-

A 600 kD

B

160 kD

1234

FIG. 3. Two major peaks of histone Hl kinase activity are detected following gel filtration of MOE prepared with NaF and ATPrS. MOE was prepared with (A) or without (B) 50 mMNaF and 2 mMATP-/S. Each extract (0.5 ml) was fractionated on an Ultrogel AcA34 column and the fractions were assayed for Hl kinase activity. Arrows indicate peak regions for 600- and 150-kDa kinases, respectively.

HI

12

FIG, 4. MPM-2 binds a 600-kDa kinase. (A) The peak fractions of the 600-kDa kinase (Lanes 1 and 2) and the 150-kDa kinase (Lanes 3 and 4) were pooled and absorbed by MPM-2 (Lanes 1 and 3) or the control antibody RDA-1 (Lanes 2 and 4). After the beads were washed, Hl kinase activity of the proteins on the beads was assayed. (B) The peak fractions of the 600-kDa kinase were pooled and absorbed by MPM-2 (Lane 1) or the control antibody RDA-1 (Lane 2) and the supernatants were assayed for Hl kinase activity.

KUANGETAL.

A

59

Novel A4Phase-Speti$c HI Kinase

8

728

7234

12

FIG. 5. The 150-kDa kinase binds to pl3-Sepharose. (A) The peak fractions of the 600-kDa kinase (Lanes 1 and 2) and the 150-kDa kinase (Lanes 3 and 4) following gel filtration were pooled and absorbed by pl3-Sepharose (Lanes 1 and 3) or control Sepharose (Lanes 2 and 4) at 4°C for 1 hr. After the beads were washed, Hl kinase activity of the proteins on the beads was assayed. (B) In this case, the 150-kDa kinase fractions were pooled and absorbed by pl3Sepharose (Lane 1) or control Sepharose (Lane Z), and the Hl kinase activity remaining in the supernatant was assayed after the beads were pelleted.

lized multiple MPM-2-reactive peptides (Fig. ‘7B). Since no MPM-2 antigens were detectable in the cyclin complex, the small amount of Hl kinase activity in the 600kDa fractions that contained cyclin B2 is probably not due to MPM-2 kinase. It is possible that the detection of a low level of cyclin-containing kinase in the 600-kDa fractions was due to incomplete separation of proteins by gel filtration in the absence of detergent. In our previous studies, we showed that MPM-2 recognized two major peptides of 58 and 180 kDa and several less abundant ones on immunoblots of mature oocyte extract (Kuang et al, 1989). Careful recalibration of the prestained molecular weight standards (see Materials and Methods) revealed that the immunoreactive peptide reported to be 58 kDa actually migrates at 72 kDa. Following gel filtration of MOE, the 72-kDa MPM-2-reactive peptide was absent in the fractions containing MPM-2 kinase activity, whereas the 180-kDa MPM-2reactive peptide was predominant (Fig. 2C). Therefore, the 180-kDa MPM-2-reactive peptide might be part of MPM-2 kinase. However, since MPM-2 kinase-positive fractions also contained other MPM-2-reactive peptides,

-

Hl

FIG. 6. Anti-cyclin B2 binds the 150-kDa kinase. The peak fractions of the BOO-kDakinase (Lanes 1 and 2) and the 150-kDa kinase (Lanes 3 and 4) were pooled and absorbed by anti-frog cyclin B2 affinity beads (Lanes 1 and 3) or control antibody affinity beads (Lanes 2 and 4) at 4°C for 1 hr. After the beads were washed, Hl kinase activity of the proteins on the beads was assayed.

-L 1 2

1234

FIG. 7. Anti-cyclin B2 antibodies bind Hl kinase activity but no detectable MPM-2 antigens. (A) MOE (100 ~1)was mixed with 20 11of protein A beads to which IgGs from anti-frog cyclin B2 immune serum (Lane 1) and preimmune serum (Lane 2) were bound. After being rotated for 2 br, the beads were pelleted and washed. Hl kinase activity of the proteins on the beads was assayed. Phosphorylation of histone Hl was revealed by autoradiography following separation of the proteins by SDS-PAGE. (B) MOE (100 ~1)was mixed with 20 pl of protein A beads to which IgGs from anti-frog cyclin B2 immune serum (Lane I), MPM-2 ascites (Lane 2), RDA-1 ascites (Lane 3), and anti-frog cyclin B2 preimmune serum (Lane 4) were immobilized as described above. After the beads were washed, proteins on the beads were released by SDS-PAGE sample buffer and separated on a 12.5% SDSPAGE for Western blot analysis for the presence of MPM-2-reactive peptides. Arrows refer to IgG heavy (H) and light (L) chains and MPM-2-reactive peptides of ‘72and 180 kDa.

further purification of MPM-2 kinase is necessary to identify the immunoreactive peptide that is part of this kinase. A Maximum Increase in MPM-2 Kinase Activity Coincides with the Activation of MPF

We examined the temporal relationship between the appearance of MPM-2 kinase and MPF activity during oocyte maturation induced by progesterone. Oocytes were treated with progesterone (1 Kg/ml) at time zero. At 30-min intervals, extracts were prepared and assayed for both MPF and MPM-2 kinase activity. To assay MPM-2 kinase activity specifically, we isolated MPM-2 kinase free of cdc2 kinase by absorbing the extract with MPM-2 affinity beads and then assayed Hl kinase activity on the beads. To quantitate MPM-2 kinase activity, we performed densitometric scanning of the bands representing phosphorylated histone Hl kinase. MPF activity was assayed by its ability to induce oocyte maturation. In immature oocytes, a low basal level of MPM-2 kinase activity was observed (Fig. 8A).

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DEVELOPMENTALBIOLOGY

VOLUME144,1991

t TImelks) MPF tMFD)

0123

-

3.5 -

Histone Hi

4

0 000446

A5

TIME (mm)

0

40 50 60 70 80 90 100 120

MPF

+----++--

FIG. 8. Maximum increase in MPM-2 kinase activity coincides with the activation of MPF. (A) Defolliculated stage VI Xenopus oocytes were incubated in MBS to which 1 pg/ml of progesterone was added at time zero. Every 30 min thereafter, 30 oocytes were collected for extract preparation. MPF activity of the extracts was assayed by microinjection into Xen0pu.s oocytes. MPM-2 kinase activity of the extracts was determined by absorbing the extract with MPM-2 affinity beads and assaying Hl kinase activity of the proteins on the beads. The time of sampling and the relative MPF activity (MFD) are indicated below each lane. (B) Extracts were prepared from dejellied unfertilized eggs (zero min) and fertilized eggs collected at different times (40-120 min) after fertilization. MPF activity and MPM-2 kinase activity were assayed as described above. The time after fertilization and the presence of MPF activity are indicated below each lane.

At 1 hr after progesterone treatment, MPM-2 kinase activity doubled and then remained constant for the next 2 hr. At 3.5 hr after exposure to progesterone, at which time MPF activity appeared in the oocytes, an additional fourfold increase in MPM-2 kinase activity occurred. We also examined the temporal relationship between the increase in MPM-2 kinase and MPF activity during the first cell cycle of fertilized eggs (Fig. 8B). Unfertilized eggs, which are arrested at the second meiotic metaphase and contain MPF activity, exhibited a high level of MPM-2 kinase activity. Forty minutes after fertilization, MPF became undetectable, indicating the entry of the eggs into interphase. Concomittantly, the activity of MPM-2 kinase became as low as 25% of its level in the unfertilized egg. Afterward, MPM-2 kinase activity showed a two-step increase as was observed during oocyte maturation. The first increase occurred prior to the detection of MPF activity (50 min after fertilization) and the second increase of MPM-2 kinase occurred at the time MPF was first detectable (80 min after fertilization), which indicates the entry of the eggs into mitosis. At 100 min after fertilization, MPF became undetectable, indicating that the eggs had entered interphase of the second cell cycle. Simultaneously, MPM-2 kinase was greatly decreased. Therefore, in both meiotic and mitotic cell cycles, there is an initial increase in MPM-2 kinase activity that precedes the activation of MPF and then a maximal increase in MPM-2 kinase activity accompanying the activation of MPF. MPM-2 Kinase Is Present in Latent Form in Immature Oocytes To determine whether the increase in MPM-2 kinase activity at M phase was due to de nova synthesis of kinase molecules or to the activation of a preexisting pro-

tein, we examined MPM-2 kinase activity during MPFinduced oocyte maturation that does not require protein synthesis. For these experiments, oocytes were injected with 70 nl of mature oocyte extract in the presence or absence of cycloheximide (100 pg/ml), a protein synthesis inhibitor. Extracts were prepared at 30-min intervals and were assayed for MPM-2 kinase and MPF activity as described above. It is clearly shown by Fig. 9 that cycloheximide did not prevent either the appearance of MPF activity or the increase in MPM-2 kinase activity. Therefore, we can conclude that the increase in MPM-2 kinase activity during oocyte maturation is mainly due to the activation of a latent form of MPM-2 kinase. MPM-2 Kinase-Positive Fractions Accelerate Progesterone-Induced Oocyte Maturation To explore the possibility that MPM-2 kinase is a positive regulator in the M phase induction pathway, we examined whether MPM-2 kinase-positive fractions had a positive effect on Xenopus oocyte maturation. First, cychhexMtfe

Hktone

--L

Hl-

llmecmilg m

+

-L

0

30 60 90 120

--+++

0

30 60 90 120

--+++

FIG. 9. Protein synthesis is not required for the increase in MPM-2 kinase activity during oocyte maturation. Defolliculated oocytes were injected with 70 nl of MOE (MFD = 4) in the presence (+) or absence (-) of cycloheximide (100 pg/ml), which was added to the MBS 20 min before the injections. At 30-min intervals, extracts were prepared from the injected ooeytes for MPF and MPM-2 kinase assays as described above. Time of sampling and MPF activity are indicated below each lane.

KUANG ET AL.

MPM-2

TABLE 1 KINASE-POSITIVE FRACTIONS ACCELERATE PROGESTERONEINDUCED OOCYTR MATURATION No. oocytes showing WSF at different times (hr) after progesterone treatment

Fraction No.

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Buffer 11 12 13 14 15 16

0 0 0 0 0 0 0

0 0 1 5 3 6 2

0 2 1 13 10 9 6

2 4 2 14 13 12 9

5 4 7 14 13 12 11

8 9 11 14 13 12 12

14 13 11 14 13 12 12

61

Novel M Phase-Specific HI Kinase

GVBD, (hr) 3.5 3.0-3.5 3.0 1.5-2.0 1.5-2.0 1.5-2.0 2.0-2.5

Note. Hl kinase-positive fractions (No. 11-16) corresponding to the 600-kDa MPM-2 kinase region obtained from AcA34 chromatography were individually concentrated 20-fold and 70 nl was injected into each of 15 oocytes per fraction. Control oocytes were injected with an equivalent volume of AcA34 column running buffer. Subsequently the oocytes were treated with 1 pg/ml progesterone and scored every 30 min for white spot formation (WSF). WSF was used to represent GVBD in these experiments since in all cases examined where WSF had occurred, GVBD had also occurred. GVBD, refers to the time required for 50% of the oocytes to undergo WSF.

we concentrated MPM-2 kinase-positive fractions (1316) as well as earlier (11 and 12) and later fractions (1’7-30) and injected them into immature oocytes. While fractions 17-30 were able to induce oocyte maturation, none of the MPM-2 kinase-positive or earlier fractions could (data not shown). Second, we injected oocytes with the MPM-2 kinase-positive and earlier fractions and subsequently treated the injected oocytes with progesterone. We found that the oocytes injected with MPM-2 kinase-positive fractions matured much earlier than the control oocytes injected with column running buffer or earlier fractions that were negative for MPM-2 kinase. For example, if 50% of the oocytes injected with buffer or fractions that contained no MPM-2 kinase matured at 3.5 hr after exposure to progesterone, oocytes injected with MPM-2 kinase-positive fractions matured within approximately half that time, i.e., 1.5-2.0 hr after progesterone treatment (Table 1). In a second experiment, the MPM-2 kinase-positive fractions were pooled and then absorbed by MPM-2 or control antibody affinity beads. When the supernatants were concentrated and microinjected into immature oocytes that subsequently were treated with progesterone, few or none of the oocytes injected with the MPM-2-absorbed sample matured precociously, whereas the majority of oocytes injected with the sample absorbed with the control antibody did mature precociously (Table 2). These results indicate that the MPM-2 kinase-positive fractions contain an MPM-2 antigen(s) that can accelerate progesterone-induced maturation.

DISCUSSION

The major findings of the present study are (1) MPM2 recognizes a kinase in Xenopus oocytes that is independent of Ca2+and cyclic AMP and phosphorylates histone Hl in vitro; (2) this kinase, designated as MPM-2 kinase, is activated during the onset of M phase; (3) MPM-2 kinase is an M phase-specific kinase distinct from the cdc2 kinase complex; and (4) MPM-2 kinase-positive fractions accelerate progesterone-induced oocyte maturation. MPM-2 kinase does not appear to be species-specific since it is also detected in mitotic HeLa cell extract (unpublished) and is recognized by MPM-2, which recognizes mitotic antigens in all species tested. Therefore, like cdc2 kinase, MPM-2 kinase meets all major criteria for a growth-associated or M phase-specific Hl kinase. Furthermore, since the percentage of Hl kinase activity contributed by MPM-2 kinase (20%) is significant, MPM-2 kinase should be considered as one of the major kinases accounting for M phase-specific Hl kinase activity. MPM-2 kinase differs significantly from cdc2 kinase in several respects. In addition to the difference in apparent molecular weight and peptide composition, MPM-2 kinase is readily inactivated during gel filtration of mature oocyte extract prepared without NaF and ATPrS, while active cdc2 kinase is recovered under the same conditions, perhaps explaining why MPM-2 kinase was not identified in previous studies. Additionally, MPM-2 kinase did not induce oocyte maturation under conditions where cd& kinase did induce oocyte maturation, suggesting that they phosphorylate different substrates in vivo. Whether MPM-2 kinase phosphorylates histone Hl at the same site as cdc2 kinase remains to be established.

MPM-2

TABLE 2 ABSORPTION ABOLISHES THE MATURATION-ACCELERATING ACTIVITY OF MPM-2 KINASE-POSITIVE FRACTIONS No. oocytes showing WSF/oocytes injected at

Samples injected

2.0 hr

2.5 hr

3.0 hr

MPM-2 absorbed RDA-1 absorbed Buffer

O/10 o/10 o/10

o/10 6/10 o/10

o/10 9/10 2/10

Note. The MPM-2 kinase-positive fractions analyzed in Table 1 were pooled and absorbed by either MPM-2 affinity beads or RDA-1 control affinity beads. Following centrifugation, the supernatants were microinjected into oocytes at 70 nl and white spot formation (WSF) was scored following stimulation of the oocytes with 1 rg/ml progesterone. The results were compared with those obtained from oocytes injected with an equivalent volume of AcA34 column running buffer.

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DEVELOPMENTALBIOLOGY

MPM-2 kinase also differs from other characterized mitotic kinases. The proto-oncogene mos encodes a kinase (Freeman et al, 1989; Singh and Arlinghaus, 1989; Roy et al, 1990) which has been shown to be a positive regulator of oocyte maturation (Sagata et ak, 1988,1989; Freeman et al., 1989). However, the mos protein is newly synthesized during maturation (Sagata et ah, 1988), whereas MPM-2 kinase preexists in immature oocytes in a latent form. NimA kinase, identified in Aspergillus nidulans (Osmani et aZ.,1988), is required for entry into mitosis. However, nimA kinase is unable to phosphorylate histone Hl in vitro (Osmani, personal communication) as does MPM-2 kinase. The niml gene in Schixosaccharomyces pombe encodes a putative kinase that is a positive regulator in the mitotic regulatory network (Russell and Nurse, 1987). We cannot compare MPM-2 kinase with niml kinase since niml kinase has not been characterized biochemically. Therefore, at this stage, we propose that MPM-2 kinase is a novel M phase-specific Hl kinase. Acceleration of progesterone-induced oocyte maturation by MPM-2 kinase-positive fractions suggests that MPM-2 kinase may be a positive regulator in the M phase induction pathway. This notion is supported by our observations that MPM-2 kinase is activated during the course of MPF activation and is stabilized by ATPrS and NaF, which also stabilize MPF activity (Lohka et aZ., 1988). It is possible that MPM-2 kinase is one of the positive regulators of MPF activation. To establish its role in M phase induction, purification of MPM-2 kinase will be attempted in our future studies. Since cd& kinase has been shown to possessboth MPF activity and M phase-specific Hl kinase activity, many laboratories use the assay of total Hl kinase activity as a measure of cd& kinase or MPF activity. Our finding that M phase-specific Hl kinase is composed of at least two major kinases suggests that great caution should be taken when total Hl kinase activity in extracts is used to represent either cd& kinase activity or MPF activity. We have shown that cdc2 kinase and MPM-2 kinase can be separated from each other by p13 absorption and MPM-2 absorption. In our previous studies, we have shown that MPM-2 immunodepletes MPF activity from mature oocyte extract (Kuang et ak, 1989). In the present study, we have clearly demonstrated that MPM-2 does not deplete cdc2 kinase activity. To explain why MPM-2 immunodepletes MPF activity but not cdc2 kinase activity, we would like to consider three possibilities. First, the Hl kinase activity of cdc2 kinase might not represent its MPF activity, i.e., for cd& kinase to have MPF activity, a further modification of the molecule might be essential which requires the presence of some MPM-2 antigen(s) in the extract. Second, some MPM-2 antigen might prevent

VOLUME144, 1991

cdc.2kinase from being inactivated upon microinjection. Therefore, much more cd&? kinase might be required to induce oocyte maturation if the MPM-2 antigen(s) is removed by immunodepletion. Finally, the MPF activity in the M phase extract might be due to two interdependent factors, cdc2 kinase and a factor recognized by MPM-2, both of which induce oocyte maturation. We have not determined which and how many of the MPM-2 antigens in mature oocyte extract contribute to MPF activity. It is possible that the MPM-2 kinase identified in the present study is one such antigen. We thank the laboratory of Paul Nurse for providing the PSTAIR antiserum, and the laboratory of Tim Hunt for the antiserum to frog cyclin B2. We also express our appreciation to the laboratories of Tim Hunt and Marc Kirschner for providing pl&Sepharose. We gratefully acknowledge Larry Etkin, Richard Behringer, Gary Gallick, and Steve Osmani for critical review of the manuscript and for insightful suggestions. This study was supported in part by Research Grants CA34783 and CA27544 (to P.N.R.) and CA-16672 from the National Cancer Institute. J.K. is supported by a Rosalie B. Hite Fellowship. REFERENCES ARION, D., MEIJER, L., BRIZUELA, L., and BEACH, D. (1988). c&Z is a component of the M phase-specific histone Hl kinase: Evidence for identity with MPF. Cell 55,371-378. BOOHER,R. N., ALFA, C. E., HYAMS,J. S., and BEACH,D. H. (1989). The protein kinase: Regulation of catafission yeast cd&/cdcl3/sucl lytic activity and nuclear localization. CeU58,485-497. BRADBURY,E. M., INGLIS, R. J., and MA~EWS, H. R. (1974). Control of cell division by very lysine rich histone (fl) phosphorylation. Nature (London) 247,257-261.

BRADBURY,E. M., INGLIS, R. J., MATTHEWS,H. R., and SARNER,N. (1973). Phosphorylation of very lysine-rich histone in Physarum polycephalum. Eur. J. Biochem. 33,131-139. BRIZUELA, L., DRAETTA, G., and BEACH, D. (1987). ~13”’ acts in the fission yeast cell division cycle as a component of the ~34~’ protein kinase. EMBO J. 6,3507-3514. BRIZUELA,L., GIIJLIO, D., and BEACH, D. (1989). Activation of the human cd& protein as a histone kinase associated with complex formation with the p62 subunit. Proc. Nat1 Acaa! Sci. USA 86,43624366. CAPONY,J. P., PICARD,A., PEAUCELLIER,G., LABBE, J. C., and DOREE, M. (1986). Changes in the activity of the maturation-promoting factor during meiotic maturation and following activation of amphibian and starfish oocytes: Their correlations with protein phosphorylation. Dev. Biol. 117, l-12. CENTONZE,V. E., and BORISY,G. G. (1990). Nucleation of microtubules from mitotic centrosomes is modulated by a phosphorylated epitope. J. Cell Sci. 95,405-411. CYERT,M. S., and KIRSCHNER,M. W. (1988). Regulation of MPF activity in vitro. Cell 53,185-195. DAVIS, F. M., and RAO, P. N. (1987). Antibodies to mitosis-specific phosphoproteins. In “Molecular Regulation of Nuclear Events in Mitosis and Meiosis” (R. A. Schlegel, M. S. Halleck, and P. N. Rao, Eds.), pp. 259-293, Academic Press, New York. DAVIS, F. M., TSAO,T. Y., FOWLER,S. K., and RAO, P. N. (1983). Monoclonal antibodies to mitotic cells. Proc. Natl. Acad. Sci. USA 80, 2926-2930.

DOREE,M., LABBE, J. C., PICARD,A. (1989). M phase promoting factor:

KUANG ET AL.

Novel M Phase-Specijc Hl Kinase

Its identification as the M phase-specific Hl histone kinase and its activation by dephosphorylation. J. Cell Sci. SuppL 12,39-51. DRAETPA,G., and BEACH, D. (1988). Activation of cd& protein kinase during mitosis in human cells: Cell cycle-dependent phosphorylation and subunit rearrangement. Cell 54,17-26. DRAETTA, G., BRIZUELA, L., POTASHKIN, J., and BEACH, D. (1987). Identification of p34 and p13 human homologs of the cell cycle regulators of fission yeast encoded by cd&+ and sueI+. Cell 50,319-325. DRAETTA, G., LUCA, F., WESTENDORF,J., BRIZUELA,L., RUDERMAN,J., and BEACH, D. (1989). cd& protein kinase is complexed with both cyclin A and B: Evidence for proteolytic inactivation of MPF. Cell 56,829-838. DRURY, K. C., and SCHORDERET-SLATKINE, S. (1975). Effects of eycloheximide on the “autocatalytic” nature of the maturation promoting factor (MPF) in oocytes of Xenopus la&s. Cell 4,269-2’74. DUNPHY,W. G., BRIZUELA,L., BEACH,D., and NEWPORT,J. (1988). The Xenopus w!& protein is a component of MPF, a cytoplasmic regulator of mitosis. Cell 54,423-431. DUNPHY, W. G., and NEWPORT,J. W. (1988a). Unraveling of mitotic control mechanisms. Cell 55,925-928. DUNPHY,W. G., and NEWPORT,J. W. (1988b). Mitosis-inducing factors are present in a latent form during interphase in the Xewpus embryo. J. Cell Biol 106.2047-2056. DUNPHY,W. G., and NEWPORT,J. W. (1989). Fission p13 blocks mitotic activation and tyrosine dephosphorylation of the Xewpas co?&protein kinase. Cell 58,181-191. ENGLE,D. B., DOONAN,J. H., and MORRIS,N. R. (1988). Cell-cycle modulation of MPM-2-specific spindle pole body phosphorylation in Aspergillus nidulans. Cell MotiL Cytoskel. 10,432-437. EVANS,T., ROSENTHAL,E. T., YOUNGBLUM,J., DISTEL, D., and HUNT, T. (1983). Cyclin: A protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division. Cell 33,389-396. FREEMAN,R. S., PICKHAM, K. M., KANKI, J. P., LEE, B. A., PENA, S. V., and DONOGHUE,D. J. (1989). Xenopus homolog of the mos proto-oneogene transforms mammalian fibroblasts and induces maturation of Xenopus oocytes. Proc. Natl. Acod Sci USA 86,5805-5809. GAUTIER,J., MATASUKAWA,T., NURSE,P., and MALLER, J. (1989). Dephosphorylation and activation of Xenopus p34* protein kinase during the cell cycle. Nature (London) 339,626-629. GAUTIER, J., MINSHULL, J., LOHKA, M., GLOTZER,M., HUNT, T., and MALLER, J. (1990). Cyclin is a component of maturation-promoting factor from Xenopus. Cell 60,487-494. GAUTIER, J., NORBURY,C., LOHKA, M., NURSE, P., and MALLER, J. (1988). Purified maturation-promoting factor contains the product of a Xenms homology of the fission yeast cell cycle control gene cd&+. Cell 54,433-439.

GERHART,J., WV, M., and KIRSCHNER,M. (1984). Cell cycle dynamics of an M phase-specific cytoplasmic factor in Xenqpus loevis oocytes and eggs. J. Cell BioL 98,1247-1255. GURDON,J. B. (1976). Injected nuclei in frog ooeytes: Fate, enlargement, and chromatin dispersal. J. Embryol. Exp. MorphoL 36,523540. HAYLES, J., BEACH,D., DURKAC~,B., and NURSE,P. (1986). The fission yeast cell cycle control gene cdc2: Isolation of a sequence SUCIthat suppresses cd& mutant function. Mol. Gen. Genet. 202,291-293. HIRAOKA, L., GOLDEN,W., and MAGNUSON,T. (1989). Spindle-pole organization during early mouse development. Dev. BioL 133,24-36. HINDLEY, J., PHEAR, G. A., STEIN, M., and BEACH, D. (1987). sucI+ encodes a predicted 13-kilodalton protein that is essential for cell viability and directly involved in the division cycle of Schizosaccharomyces pombe. MoL Cell. Biol. 7,504-511.

KARSEN~, E., BRAVO,R., and KIRSCHNER,M. (1987). Phosphorylation changes associated with the early cell cycle in Xenopus eggs. Dev. BioL 119,442-453.

63

KUANG, J. K., ZHAO, J., WRIGHT, D. A., SAUNDERS,G. F., and RAO, P. N. (1989). Mitosis-specific monoclonal antibody MPM-2 inhibits Xerwpus oocyte maturation and depletes maturation-promoting activity. Proc. NatL Acad. Sci. USA 86,4982-4986. LABBE, J. C., PICARD,A., KARSENTI,E., and DOREE,M. (1988a). An M phase-specific protein kinase of Xenopas oocytes: Partial purification and possible mechanism of its periodic activation. Dev. Biol. 127,157-169. LABBE,J. C., LEE, M. G., NURSE,P., PICARD,A., and DOREE,M. (1988b). Activation at M phase of a protein kinase encoded by a starfish homologue of the cell cycle control gene co!&+. Nature (London) 335, 251-254. LABBE,J. C., PICARD,A., PEAUCELLIER,G., CAVADORE,J. C., NURSE,P., and DOREE,M. (1989). Purification of MPF from starfish: Identification as the Hl histone kinase p34&‘and a possible mechanism for its periodic activation. Cell 54, 253-263. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227,680685. LAKE, R. S. (1973). Further characterization of the Fl-histone phosphokinase of metaphase-arrested animal cells. J. Cell Biol. 58,317331. LAKE, R. S., and SALZMAN,N. P. (1972). Occurrence and properties of a chromatin-associated Fl-histone phosphokinase in mitotic Chinese hamster cells. Biochemistry 11,4817-4825. LANGAN,T. A., GAUTIER,J., LOHKA, M., HOLLINGSWORTH, R., MORENO, S., NURSE,P., MALLER,J., and SCALAFANI,R. A. (1989). Mammalian growth-associated Hl histone kinase: Homolog of cdc2+/cdc28 protein kinases controlling mitotic entry in yeast and frog cells. Mol. CelL BioL 9, 3860-3868.

LEE, M. G., and NURSE, P. (1987). Complementation used to clone a human homologue of the fission yeast cell cycle control gene c&2. Nature

(London) 327,31-35.

LOHKA, M. J. (1989). Mitotic control by metaphase-promoting factor and cdc proteins. J. Cell Sci. 92,131-135. LOHKA, M. J., HAYES, M. K., and MALLER, J. L. (1988). Purification of maturation-promoting factor, an intracellular regulator of early mitotic events. Proc. Natl. Acod Sci. USA 85, 3009-3013. LOHKA, M. J., KYES, J. L., and MALLER, J. L. (1987). Metaphase protein phosphorylation in Xenopus laevis. MoL Cell. BioL 7,760-768. MARKS, D. B., PAIK, W. K., and BORUN,T. W. (1973). The relationship of histone phosphorylation to deoxyribonucleic acid replication and mitosis during the HeLa S3 cell cycle., J. BioL Chem. 248,5660-5667. MASUI, Y., and MARKERT,C. L. (1971). Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. J. Exp. ZooL 177,129-146. MATTHEWS,H. E., and HUEBNER, V. D. (1985). Nuclear protein kinases. Mol. Cell. Biochem. 59,81-89. MELTER,L., ARION, D., GOLSTEYN,R., PINES,J., BRIZUELA,L., HLJNT,T., and BEACH, D. (1989). Cyclin is a component of the sea urchin M phase specific histone Hl kinase. EMBO. J. 8,2275-2282. MINSHULL, J., BLOW,J. J., and HUNT, T. (1989). Translation of cyclin mRNA is necessary for extracts of activated Xenopus eggs to enter mitosis. Cell 56, 947-956. MURRAY, A. W. (1988). A mitotic inducer matures. Nature (Lolzdon) 335,207-208. MURRAY,A. W., and KIRSCHNER,M. W. (1989). Cyelin synthesis drives the early embryonic cell cycle. Nature (London) 339,275-280. MURRAY, A. W., SOU)MON,M. J., and KIRSCHNER,M. W. (1989). The role of cyclin synthesis and degradation in the control of maturation promoting activity. Nature (London) 339, 280-286. NURSE,P. (1990). Universal control mechanism regulating onset of M phase. Nature (London) 344,503-508.

64

DEVELOPMENTALBIOLOGY

NURSE,P., and THURIAUX, P. (1980). Regulatory genes controlling the initiation of mitosis. Genetics 96,627-637. NURSE,P., THURIAUX, P., and NASMYTH, K. (1976). Genetic control of cell division cycle in the fission yeast Schizosaccharmyces pmnbe. MoL Gen. Genet. 146,167-178. OSMANI, S. A., Pu, R. T., MORRIS,N. R. (1988). Mitotic induction and maintenance by overexpression of a GZ-specific gene that encodes a potential protein kinase. Cell 53,237~244. PICARD, A., LABBE, J. C., PEAUCELLIER, G., LE BOUFFANT, F., LE PEUCH, C. J., and DOREE, M. (1987). Changes in the activity of the maturation-promoting factor are correlated with those of a major cyclic AMP and calcium independent protein kinase during the first mitotic cell cycles in the early starfish embryo. Dew. Growth D&w. 29,93-103. PONDAVEN,P., MEIJER, L., and BEACH, D. (1990). Activation of Mphase-specific Hl kinase by modification of the phosphorylation of its p34* and cyclin components. Genes Dev. 4,9-17. RAO, P. N., ZHAO,J-Y., GANJU, R. K., and ASHORN,C. L. (1989). Monoclonal antibody against the centrosome. J. Cell Sci. 93, 63-69. ROY, L. M., SINGH, B., GAUTIER, J., ARLINGHAUS, R. B., NORDEEN, S. K., and MWER, J. L. (1990). The cyclin B2 component of MPF is a substrate for the c-mop proto-oncogene product. Cell 61,825-831. RUSSEL,P., and NURSE,P. (1987). The mitotic inducer nim l+ functions in a regulatory network of protein kinase homologs controlling the initiation of mitosis. Cell 49, 569-576. SAGATA, N., DAAR, I., OSKARSSON,M., SHOWALTER,S. D., and VANDE WOUDE,G. F. (1989). The product of the mos proto-oncogene as candidate “initiator” for oocyte maturation. Science 245,64f3-646. SAGATA, N., OSKARSSON,M., COPELAND, T., BRUMBAUGH, J., and VANDE WOUDE,G. F. (1988). Function of c-mos proto-oncogene product in meiotic maturation in Xenw oocytes. Nature (London) 335, 519-525.

SIMANIS,V., and NURSE,P. (1986). The cell cycle control gene cd&+ of fission yeast encodes a protein kinase potentially regulated by phosphorylation. Cell 45,261-268. SINGH, B., and ARLINGHAUS, R. B. (1989). Vimentin phosphorylation by ~37”“” protein kinase in vitro and generation of a 50 kDa cleavage product in v-nws transformed cells. Virology 173,144-156.

VOLUME 144,1991

SMITH, L. D., and ECKER,R. E. (1971). The interaction of steroids with Rana pipiens oocytes in the induction of maturation. Dev. Biol. 25, 232-247. SIJNKARA,P. S., WRIGHT, D. A., and RAO, P. N. (1979). Mitotic factors from mammalian cells induce germinal vesicle breakdown and chromosome condensation in amphibian oocytes. Proc. Natl Acad. Sci USA 76,2799-2802.

SWENSON,K. I., FARRELL,K. M., and RUDERMAN,J. V. (1986). The clam embryo protein cyclin A induces entry into M phase and the resumption of meiosis in Xenopus oocytes. CeU47,861~870. VANDRE, D. D., and BORISY,G. G. (1989). Anaphase onset and dephosphorylation of mitotic phosphoproteins occur concomitantly. J. Cell Sci. 94,245-258.

VANDRE, D. D., DAVIS, F. M., RAO, P. N., and BORISY,G. G. (1984). Phosphoproteins are components of mitotic microtubule organizing centers. Proc. Natl. Aced Sci. USA 81,4439-4443. VANDRE, D. D., DAVIS, F. M., RAO, P. N., and BORISY, G. G. (1986). Distribution of cytoskeletal proteins sharing a conserved phosphorylated epitope. Eur. J. Cell Biol. 41, 72-81. WASSERMAN,W. J., and MASUI, Y. (1975). Effects of cycloheximide on a cytoplasmic factor initiating meiotic maturation in Xenopus oocytes. Exp. Cell Res. 91, 381-392. WASSERMAN,W. J., and SMITH, L. D. (1978). The cyclic behavior of a cytoplasmic factor controlling nuclear membrane breakdown. J. Cell Biol. 78, R15-R22. WOODFORD,T. A., and PARDEE, A. B. (1986). Histone Hl kinase in exponential and synchronous populations of Chinese hamster fibroblasts. J. Biol. Chem. 261,4669-4676. Wu, M., and GERHART,J. C. (1980). Partial purification and characterization of the maturation-promoting factor from eggs of Xenspus laevis. Dev. Biol. 79,455-477. ZHAO,J., KUANG, J., ADLAKHA, R. C., and RAO, P. N.. (1989) Threonine phosphorylation is associated with mitosis in HeLa cells. FEBSLetters 249,389-395.

ZEILIG, C. E., and LANGAN, T. A. (1980). Studies on the mechanism of activation of mitotic histone Hl kinase. Biochem. Biophys. Res. Commun. 95.1372-1379.