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Regulation of pl05"~~' and p34cdc2during meiosis in Schizosaccharomyces pombe MALEKIDAYA-MAKIN Biomedical Research Centre, University of British Columbia, Vancouver, B.C., Canada V6T 123

PHILIPPESZANKASI Fred Hutchinson Cancer Research Centre, Seattle, WA 98104, U.S.A.

LIRENTANG Department of Zoology, University of British Columbia, Vancouver, B.C., Canada V6T 123

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DIANAMACRAE Biomedical Research Centre, University of British Columbia, Vancouver, B.C., Canada V6T 123 AND

STEVEN L. PELECH' Biomedical Research Centre and Department of Medicine, University of British Columbia, Vancouver, B. C., Canada V6T 123 and Kinetek Biotechnology Corporation, 7600 No. 1 Road, Richmond, B.C., Canada V7C 1T6 Received September 13, 1991 P., TANG,L., MAC-, D., and PELECH,S. L. 1992. Regulation of pl~5"' and p34cdc2 DAYA-IV~AKIN, M., SZANKASI, during meiosis in Schizosaccharomyces pombe. Biochem. Cell Biol. 70: 1088-1096. Temperature-sensitive patl mutants of the fission yeast Schizosaccharomycespombecan be induced to undergo meiosis at the restrictive temperature, irrespective of the matl configuration and the nutritional conditions. Using a combination of exit from stationary phase and thermal inactivation of the 52-kilodalton protein kinase that is encoded by the patl (also called ranl) gene, highly synchronous meiotic cultures were obtained. Synthesis and tyrosyl phosphorylation of ~ 3 4 ' ~was ' ~ evident during meiotic G,and S phases. During this period there was increased expression of pl~5wee', a protein kinase implicated in the tyrosyl phosphorylation of p34cdc2.Following a relatively brief G, period, during which a reduction in the steady-state level of p105wee' occurred, there was an approximately 19-fold increase in the histone H1 phosphotransferase activity of p34cdc2.Only a single peak of histone H1 kinase activation was observed, which implies that unlike meiosis in amphibians and echinoderms, p34'dc2 is functional only during one of the meiotic divisions in S. pombe, presumably meiosis 11. Stimulation of the kinase activity of p34cdc2was associated with its tyrosyl dephosphorylation. This is analogous to mitotic M phase and suggests parallels in the mechanism of activation of ~ 3 4 ' ~during ' ~ mitosis and one of the meiotic divisions in S. pombe. Key words: weel, cdc2, ranl, cell cycle, meiosis. and ~ 3 4 ~ ~ ' ~ DAYA-MAKIN, M., SZANKASI, P., TANG,L., MAC-, D., et PELECH,S. L. 1992. Regulation of pl05 during meiosis in Schizosaccharomyces pombe. Biochem. Cell Biol. 70 : 1088-1096. Chez les mutants patl, sensibles a la temperature de la levure scissipare Schizosaccharomycespombe, il est possible d'induire la mtiose a la temptrature de restriction, indtpendamment de la configuration matl et des conditions nutritionnelles. Utilisant une combinaison permettant la sortie de la phase stationnaire et l'inactivation thermique de la prottine kinase de 52 kilodaltons, codbe par le gene patl (tgalement appelt ranl), nous avons obtenu des cultures mtiotiques hautement synchrones. La synthtse et la tyrosyl phosphorylation de la ~ 3 4 ' ~sont ' ~ tvidentes durant les phases G, et S de la mtiose. Durant cette ptriode, il y a expression accrue de la p105w"', une prottine kinase impliqute dans la tyrosyl phosphorylation de la p34cdc2.Suite ti une pCriode G, relativement breve, durant laquelle se produit une reduction du taux a l'tquilibre de la p105wee1,l'activitt histone H1 phosphotransftrasique de la p34cdc2augmente d'environ 19 fois. Nous n'avons observt qu'un seul pic d'activation de l'histone H1 kinase; cela implique, qu'a la difference de la mtiose chez les amphibiens et les tchinodermes, la p34'dc2 n'est fonctionnelle que durant l'une des divisions mtiotiques, probablement la mtiose 11. La stimulation de l'activitt kinasique de la p34'dc2 est associte a sa tyrosyl dtphosphorylation. Cela est analogue ii la phase mitotique M et sugg2re des comparaisons dans le micanisme d'activation de la p34cdc2durant la mitose et l'une des divisions mtiotiques chez S. pombe. Mots cl6s : weel, cdc2, ranl, cycle cellulaire, mtiose. [Traduit par la rtdaction]

Introduction Genetic studies with Schizosaccharomycespombe have uncovered a network of protein kinases, including p34cdc2, ABBREVIATIONS: MPF, maturation promoting factor; TCA trichloroacetic acid; DAPI, 4,&dimidino-2-phenylindole; MOPS, morpholinopropanesulfonic acid; SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline; kDa, kilodalton; ts, temperature sensitive: ELISA.. enzyme-linked immunosorbent assay. ' ~ b t h o rto whom all correspondence should be iddressed. Primed In Canada / lmpr~meau Canada

plOSwee', p66mik,and p67"'"', that appear to coordinate cell division with growth (Nurse and Thuriaux 1977; Fantes and Nurse 1978; Fantes 1979, 1981; Nurse and Bissett 1981; Russell and Nurse 1987a. 1987b) (reviewed in Nurse 1990; Pelech et al. 1990). ~iochemicalstudies have demonstrated that ~ 3 4 is~a ~protein-sewthreonyl ~ ' kinase, which is activated by dephosphorylation of TY~-15 in the presumptive ATP-binding domain (Simanis and Nurse 1986; Gould and Nurse 1989) and phosphorylation of Thr-167 (Gould et al. 1991). The CDNA sequence of ~ 1 0 5 " ~resembles ~' that of

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DAYA-MAKIN ET AL.

protein-seryl/threonyl kinases (Russell and Nurse 1987a), but the overexpressed protein in either insect cells or yeast also undergoes autophosphorylation on tyrosyl residues and can phosphorylate an exogenous substrate such as angiotensin on tyrosine (Featherstone and Russell 1991; Parker et al. 1991). Overproduction of pl05 inhibits S. pombe mitosis in a dosage- and nutrient-dependent manner (Nurse 1975; Nurse and Thuriaux 1977; Fantes and Nurse 1978; Fantes 1979, 1981; Russell and Nurse 1987a). Since dominant alleles of cdc2 exhibit insensitivity to inhibition of mitosis by wee1 +,plOSweel appears to act upstream in a kinase cascade to negatively modulate ~ 3 4 ' ~ activity '~ (Fantes 1981; Nurse and Thuriaux 1977). However, no change in the tyrosyl phosphorylation state of ~ 3 4 ' ~has '~ been detected in wee1 - mutant strains (Gould et al. 1990), which implies that the action of p l 0 5 ~ is ~ redundant ~' with that of another protein-tyrosyl kinase. ~ 6 6 exhibits ~ ' ~a high degree of homology with ~ 1 0 5 (Lundgren ~ ~ ~ ' et al. 1991). In double mutants defective in both mikl and wee1 functions, p34cdc2 rapidly loses phosphate on tyrosine, indicating that either p66m'k or pl~5wee'can promote tyrosyl phosphorylation of p34'd'2 (Lundgren et al. 1991). Neither protein kinase has been shown to directly phosphorylate ~ 3 4 ' ~in' ~vitro. However, co-overexpression of pl~5wee'and ~ 3 4 ' ~in' ~a baculovirus system resulted in a low level of tyrosyl phosphorylation of p34'd'2. Since this reaction was enhanced dramatically when either cyclin A or B was also overexpressed in this system, cyclins appear to play a critical role in directing tyrosyl phosphorylation of ~ 3 4 ' ~(Parker '~ et al. 1991). The inhibitory action of p l 0 5 ~ on ~ ~ p34' ' d'2 activity is counteracted by ~ 8 0 ' ~(Russell ' ~ ~ and Nurse 1986) and by another protein kinase, ~ 6 7 " ' ~(also ' called p67Cdr' (Feilotter et al. 1990). The protein level of ~ 8 0 ' ~peaks '~~ near the onset of M phase in S. pombe (Ducommun et al. 1990). p8ocdCZ5 has recently been identified as a member of a new class of protein-tyrosyl phosphatases and it can dephosphorylate Tyr-15 of p34'd'2 in vitro (Moreno and Nurse 1991; Strausfeld et al. 1991; Gautier et al. 1991; Dunphy and Kumagai 1991). Increased niml expression rescues mutants lacking cdc25 and advances cells into mitosis at a reduced size; loss of niml delays mitosis until cells have attained a larger size. An attractive, but untested, hypothesis is that ~ 6 7 " ' ~phosphorylates ' and inactivates pl05 A p34'd'2-cyclin B complex has been identified as the substance known as MPF, which induces precocious resumption of meiotic maturation when microinjected into echinoderm and amphibian oocytes, which are naturally blocked at prophase of meiosis I (Arion et al. 1988; Draetta et al. 1989; Dunphy et al. 1988; Gautier et al. 1988; Labbe et al. 1988a, 1988b; Meijer et al. 1989). Hormonal induction of oocyte maturation triggers the tyrosyl dephosphorylation and activation of ~ 3 4 ' ~ ' The ~ . biochemistry of activation and regulation of ~ 3 4 ' ~during ' ~ meiotic differentiation in S. pombe has not been studied. In temperature-sensitive pat (ranl) mutants of S. pombe, vegetative cells switch to a meiotic differentiation program at restrictive temperatures (Iino and Yamamoto 1985; Nurse 1985). Using thermal induction of meiosis and the appropriate culture conditions, we have obtained highly synchronous meiotic cultures which have enabled us to analyze p105 and p34'dc2 during the course of meiosis in S. pombe.

Materials and methods Yeast strains and culture conditions Strains of S. pombe, designated GP48 (h+ endl-458 patl-114) and the stable diploid G P 333 ( h + ade6-m210 ura4-294 patl-114/h+ade6-m375 leul-32 argl-2 patl-114 endl-158) were used (Iino and Yamamoto 1985; Nurse 1985). For the induction of meiosis, the cultures were grown in YEL (Gutz et al. 1974) at 2S°C to stationary phase, and the cells were then collected by centrifugation, washed, and transferred to modified EMM2 (Nurse 1975) medium at a density of 0.1 A , units. The cultures were further incubated (14-16 h) at 2S°C to a density of 0.3 units. The temperature of the cultures was rapidly raised to 33°C by immersion in hot water and constant agitation (approximately 3 min). Preparation of yeast extracts At various intervals after the induction of meiosis, the cells were lysed by shearing in a bead beater, in a solution of 50 mM TrisHCl (pH 7.5),500 mM NaCI, 1 mM EDTA, 2 mM EGTA, 25 mM 0-glycerol phosphate, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 50 pM phenylmethanesulfonyl fluoride, 20 pg/mL 1-chloro-3-tosylamido-7-amino-L-2-heptanone, and 0.5 pg/mL each of aprotinin, leupeptin, and pepstatin. The lysate was centrifuged at 100 000 x g for 20 min at 4OC. Labelling of cells with [31ilJluracil Following temperature induction, as described above, 3 p ~ i [ ~ ~ ] u r awas c i ladded to a 25 mL culture. Duplicate cultures were also treated with 50 mM hydroxyurea 30 min later. At various time points, 5 pg of calf thymus DNA and 10 mL of 10% TCA with 1 mg/mL of uracil were added to 2 mL of the cultures. Following 15 min of incubation on ice, the samples were centrifuged for 10 min at 12 000 x g at 4°C. The supernatant was drained, resuspended in 1 mL of 0.6 M NaOH with 1 mg/mL of uracil, and heated at 6S°C for 2 h. The samples were chilled prior to the addition of 1 mL of 0.6 M HCI and 20% TCA. After 20 min of incubation on ice, the precipitate was collected by filtration through GC/F filters and counted with scintillation fluid. Fluorescence microscopy Cells (10 mL of the culture) were harvested at 1-h intervals following temperature shift up as described above. Staining with DAPI was performed essentially as described by Pringle et al. (1989). The asci were scored for the number of spores by fluorescence microscopy. Production of antibodies Polyclonal antibodies were raised in rabbits against peptides which were synthesized using an Applied Biosystems 430A automated peptide synthesizer. The sequences of the peptides were as follows. ( i ) NTSSHRSYGLRRGDQMMEDNQVNVGC (weel-T) was designed to represent a highly truncated form of plOSweel.The weel-T peptide featured amino acid residues 6-17, followed by the last 12 C-terminal residues predicted from the nucleotide sequence of wee1 (Russell and Nurse 1987~).( i i ) PKERNRLLQEVSIQRALKGHDHIVELMDSWEHGGC (weel-111) was a sequence in subdomain I11 in the wee1 gene (Hanks et al. 1988). (iii)EGVPSTAIREISLLKE (PSTAIRE) was based upon a highly conserved region in p34cdc2homologs from other species (Nurse 1990). All of these antibodies are now commercially available (Upstate Biotechnology Inc., Lake Placid, N.Y.). Rabbit polyclonal antisera against recombinant baculoviralexpressed S. pombe pl~5wee',as well as p105wee', were kindly provided by Drs. Helen Piwnica-Worms and Laura Parker (Tufts University, Boston, Mass). +

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Sucrose density gradient centrifugation Sucrose density gradient centrifugation was performed as previously described (Ludlow et al. 1990). Schizosaccharomyces pombe (GP 48) extracts (approximately 8 mg) were layered on

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5-30% sucrose gradients prepared in a solution of 50 mM Tris-HC1 (pH 8.0), 120 mM NaCl, 0.5% Nonidet P-40, 5 mM NaF, and 200 pM sodium orthovanadate, supplemented with 10 pg/mL each of aprotinin, phenylmethylsulfonyl fluoride, and leupeptin. The gradients were centrifuged for 4.5 h at 154 800 x g at 4°C in a Sorvall TH641 rotor. Fractions were collected from the top using a Gilson fraction collector with approximately 160 pL/fraction. Fifty microlitres of odd numbered fractions were mixed 1:l with Laemmli's sample buffer for Western blotting analysis.

Gel filtration chromatography on Superose 6 Two milligrams of S. pombe extract protein in 500 pL was applied to a Superose 6 (Pharmacia) column equilibrated in a mixture of 10 rnM MOPS, 25 mM P-glycerophosphate, 2 mM EDTA, 5 mM EGTA, 2 mM sodium orthovandate, and 150 mM NaCl. The column was developed in a fast liquid chromatography system (Pharmacia) with the same buffer at a flow rate of 15 mL/h and 250-pL fractions were collected. Chromatography on p13suc'-~epharose Schizosaccharomycespombe extracts (4 mg protein), prepared as described above, were incubated with 300 pL packed volume of p13mC1(purified from recombinant Escherichia coli (Brizuela et al. 1987)) linked to CNBr-activated Sepharose CL4B, at 4OC for 3 h with rotation. The beads were washed three times with bead buffer (Meijer et al. 1989) and eluted with Laemmli's SDS sample buffer (Laernmli 1970) for analysis on Western blots. The histone H1 phosphotransferase activity of ~ 3 4 ' ~ was " assayed directly on p13suc'-~epharosewithout elution of the enzyme, essentially as previously described (Meijer et al. 1989). Immunoblotting Recombinant plO5 and S. pombe extracts (crude or fractions from p13suc1-~epharose or Superose 6 chromatographies or from sucrose gradients) were resolved on 10 or 15% SDSpolyacrylamide gels and transferred to nitrocellulose (Towbin et al. 1979), using the Bio-Rad transblot apparatus at 8OC for 4 h at 100 V. The blots were probed with rabbit anti-peptide antibodies (following affinity purification on a peptide column) or mouse monoclonal anti-phosphotyrosine antibodies (PY-20, ICN). Alkaline phosphatase conjugated goat anti-rabbit or goat antimouse antibodies were used for visualization as specified by the supplier (Bio-Rad). Enzyme-linked immunosorbent assay for pl05"~~' Fifty microlitres of selected fractions following Superose 6 chromatography of S. pombe extracts were coated on to wells of Microtest I11 Flexible Assay plates (Falcon). After incubation for 2 h, the plates were washed with TBS containing 0.5% skim milk. Fifty microlitres of 1:1000 dilution of affinity purified weel-T antibody (in TBS and 0.5% skim milk) were added and the plate was further incubated at 4°C for 2 h. Following extensive washing with TBS and 0.5% skim milk, 50 pL of 1:2000 dilution goat antirabbit conjugated to horseradish peroxidase was added. After 2 h at room temperature, the plates were washed and 50 pL of a substrate solution was added (0.5 mg/mL of 2,2'-azinodi(3-ethylbenzthiazoline)sulfonicacid diammonium salt in 0.06 M citric acid - 0.1 M Na2HP0,.H20 (pH 4.5) plus H20, to a final concentration of 0.006%). The colorimetric reaction was analyzed using an autoreader (Bio-Tek Instruments).

Results Induction of synchronous meiotic cultures of S. pombe To obtain synchronous meiotic cultures of S. pombe, we have used exit from stationary phase, followed by temperature induction of meiosis in the temperature-sensitivepat1 (ranl) mutants (Iino and Yamamoto 1985; Nurse 1985). The pat1 gene encodes a 52-kDa putative protein-seryl/threonyl kinase that inhibits entry into meiosis (McLeod and Beach

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FIG. 1. Synchronized meiosis in S. pornbepatl-114. Incorporation of ['~luracil in DNA was evaluated in GP 48 (A) and GP 333 (0) following thermal induction of meiosis. The graph indicates the duration of meiotic replication, which was abolished in cells treated with hydroxyurea (HU) ( 0 ) .Cells were also stained with DAPI for fluorescence microscopy during the course of meiosis. "X" represents the percentage of cells which formed asci with four nuclei (GP 333). Synchronous sporulation occurred between 5.5 and 6 h after the induction of meiosis.

1986, 1988). Thus, upon thermal inactivation of this kinase in cells which carry the ts mutation, vegetative cells initiate meiosis in the absence of nutritional deprivation (Iino and Yarnamoto 1985; Nurse 1985) and with a high degree of synchrony under the culture conditions used in our experiments. This is illustrated in Fig. 1. Shortly after temperature shift up, DNA synthesis was initiated in both the diploid GP 333 and haploid GP 48 strains. Meiotic S phase was abolished in parallel cultures, which were treated with hydroxyurea (an inhibitor of DNA synthesis). The pattern of incorporation of [3~]uracilin DNA showed that the cells entered meiotic S phase with a high degree of synchrony. This was clearly confirmed by analysis of the timing of sporulation by DNA staining with DAPI. In both GP 333 and GP 48, 90% sporulation ocurred between 5.5 and 6 h following temperature shift up. Asci with four nuclei were clearly visible in the diploid cells (GP 333). In cultures of GP 48, abnormal asci were seen, which was a consequence of reductive meiotic divisions in a haploid state (Iino and Yamamoto 1985; Nurse 1985). Zmmunodetection of pl Ojweel during meiosis in S. pombe To characterize the gene product of wee1 in S. pombe, we raised polyclonal antibodies in rabbits against two synthetic peptides, one based upon the predicted amino and carboxy termini of wee1 (anti-weel-T) and the other patterned after the kinase subdomain I11 region (Hanks et al. 1988) of wee1 (anti-weel-111). These antibodies were affinity purified on peptide-agarose columns to increase their specificity. Both antibodies permitted detection of recombinant baculovirus-expressed pl05 on immunoblots, with the anti-weel-T antibody displaying greater sensitivity than the anti-weel-I11 antibody (Fig. 2). The smear of lower molecular mass bands that were visualized with the antiweel-T antibody were also evident with rabbit antisera devel+

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FIG. 2. Irnmunodetection of recombinant baculovirus-

expressed p105 Extracts from sf9 cells that expressed recombinant S. pombe p105wee1were prepared as described (Parker et al. 1991).Approximately 100 pg of total cell protein in each lane was subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting with polyclonal rabbit antibodies raised against purified recombinant (A), weel-T peptide (B), and weel-111 peptide (C). The arrow points to a highly immunoreactive protein of M, 105 000 that corresponds to p105wee'. This protein was not detected with any of these antibodies in extracts from control sf9 cells that were not transfected with S. pombe wee1 (data not shown). Migration positions of the prestained molecular mass marker proteins phosphorylase b (110 kDa), bovine serum albumin (84 kDa), ovalbumin (47 kDa), and carbonic anhydrase (33 kDa) are evident to the left of each panel.

oped against the baculovirus-expressed p105wee1 and appeared to be a consequence of the susceptibility of the recombinant protein to proteolysis (Fig. 2). The specificity of anti-weel-T and anti-weel-I11 was supported by the failure of these antibodies to detect a 105-kDa protein in immunoblots of extracts from a wee1 deletion mutant of S. pombe (Featherstone and Russell 1991) and in extracts from sf9 insect cells that did not express recombinant pl05 (data not shown). When extracts from cultures of GP 333 or G P 48 that had been induced t o undergo meiosis were probed in immunoblots with anti-weel-T antibodies, only one prominent immunoreactive protein of 105 kDa, the size expected for the wee1 + gene product, was visualized (Figs. 3A and 3B). This protein was also detected more weakly when the extracts were probed with the anti-weel-I11 antibody (data not shown). This immunoreactivity with two distinct preparations of antibodies supported the assignment of the 105-kDa protein in the patl ts strains as the wee1 gene product. The quantity of ~ 1 0 5 in~ the ~ ~ haploid ' stain G P 48 was much higher than in the diploid G P 333 (Figs. 3A and 3B) and wild-type strains (data not shown) of S. pombe. In extracts from both G P 333 and G P 48, p l ~ 5 accumu-

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FIG. 3. Immunodetection of pl~5w"' during meiosis in S. pombe. GP 48 (A) or GP 333 (B) extracts from mitotic cultures (lane 1,O h) or from cultures that were induced to undergo meiosis (lanes 2-7, 1-6 h) were probed in Western blots with anti-weel-T peptide antibodies. The arrow points to a highly immunoreactive protein of M, 105 000 that was maximally expressed by 2 h postinduction. The level of pl~5wee1 was greater in GP 48 (A) than GP 333 (B). In both strains p l ~ 5 accumulated during the early stages of meiosis, decreased as the cells approached M phase (3-4 h), and was undetectable during sporulation (5.5-6 h). Migration positions of the prestained molecular mass marker proteins phosphorylase b (110 kDa), bovine serum albumin (84 kDa), ovalbumin (47 kDa), carbonic anhydrase (33 kDa), and soybean trypsin inhibitor (28 kDa) are indicated on the left.

lated during the early stages of meiosis and reached a maximum level at approximately 2 h postinduction, which was coincident with the initiation of DNA replication. Following this stage, there was an apparent decrease in the amount of p105 detectable on Western blots (in extracts from both GP 48 and G P 333), and the protein was undetectable as the cells approached meiotic M phase. (Fig. 3).

Synthesis and tyrosylphosphorylation and dephosphorylation of p34cdc2during meiosis in patl ts mutants of S . pombe During mitosis in the fission yeast, p34cdc2is complexed with 13- and 56-kDa proteins specified by the sucl and cdcl3 genes, respectively (Booher and Beach 1987; Booher et al. 1989; Brizuela et al. 1987; Hayles et al. 1986; Hindley et al. 1987). Agarose beads to which p13SuC1had been covalently linked facilitated the depletion of essentially all of the PSTAIRE immunoreactivity of p34cdc2 from G P 48 and GP 333 cell extracts (data not shown). We exploited the high affinity of p34cdc2 for p13suc1-agarose for the selective assay of the histone H1 phosphotransferase activity of p34cdc2(Brizuela et al. 1987; Meijer et al. 1989). These analyses indicated that 2-3 h following commitment to meiosis, there was an increase in the level of p34cdc2protein and it was phosphorylated on tyrosine (Fig. 4). This was evident by the increase in anti-phosphotyrosine immuno-

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FIG.4. Synthesis and tyrosine phosphorylation of p34cdc2during meiosis in S. pombe patl-114. Immunoblot of p13SuC'-~epharose-adsorbed proteins with affinity purified rabbit anti-PSTAIRE antibodies (A, GP 48; B, GP 333) or mouse monoclonal PY-20 anti-phosphotyrosineantibodies (C, GP 333). The arrow indicates p34cdc2,which was detectable with antiPSTAIRE throughout meiosis in GP 333 (B) and up to 4 h posttemperature shift in GP 48 (A). Two species of p34cdc2were resolved in GP 48 at 2 h (A, lane 3). At 5 h there was reduced ~ ' ' antiphosphotyrosine antibodies immunoreactivity of ~ 3 4 ~ with (C, lane 6). reactivity in Western blots (Fig. 4C) and it indicated that the protein-tyrosyl kinase that phosphorylated p34cdc2was active during these early stages of meiosis. When extracts from the haploid cells were probed with the anti-PSTAIRE antibody, two species of p34cdc2at 2 h postmeiotic induction were detected (Fig. 4A). The faster migrating species did not immunoreact with anti-phosphotyrosine antibodies (data not shown). The possibility that the faster migrating species of p34cdc2was due to a population of cells which were still in the previous mitotic M phase was excluded because phosphotransferase activity toward histone H I , a known substrate of ~ 3 4 ~ ~ ' was ' , not associated with it (Fig. 5). It is presently unclear if there is any catalytic activity associated with this tyrosine dephosphorylated form, because the substrates for pre-M-phase p34cdc2are not known. In GP 48, and not in GP 333, degradation of ~ 3 4 was ~ ~evident ' ~ following its activation at the meiotic M phase. This may be a consequence of the catastrophic meiosis which occurs in these haploid cells. Although p34cdc2was not detectable by immunoblotting analysis in extracts prepared from 5-h postmeiotic induction of GP 48, some histone H1 phosphotransferase activity was measurable. This may reflect the presence of highly activated p34cdc2that was below the threshold level of detection on Western blots. Kinase activation of p34cdc2during meiotic M phase in pat 1-114 ts-mutants Upon shifting the cultures to the restrictive temperature, there was an apparent reduction in p13suc1-associated histone H1 phosphotransferase activity of p34cdc2as the cells became synchronized, presumably in GI (Fig. 5). This was followed by a dramatic 19-fold increase in its kinase activity that peaked at 4-4.5 h postinduction and then by

Time at 33OC (h)

FIG. 5. Analysis of the histone H1 kinase activity of ~ 3 4 ~ ~ ~ ' kinase during meiosis in S. pombe patl-114. The histone H1 phosphotransferase activity was measured following p13suc'-~epharose affinity chromatography of p34ed'zin extracts from GP 48 (0 and GP 333 (A) cells. The histone H1 kinase activity of p34' " initially decreased after 1 h and then increased to peak at 4-4.5 h following a shift of the cultures to 33OC, and this marks the timing of meiotic M phase.

1

a marked decrease by 6 h, at which point spores could be visualized by microscopy (data not shown). Notably a single peak in p34cdc2 kinase activity was discernable, which presumably occurred at the second meiotic division. (See Discussion. DAPI staining was not useful in distinguishing the transition from meiosis I to meiosis 11. The least ambiguous images were not observed until after the completion of the second meiotic division. At this point four evenly spaced out nuclei were seen in each asci of GP 333.) In a previous study (Gould and Nurse 1989), phosphoamino acid analysis of p34cdc2immunoprecipitated from [32~]phosphate-labelledcells that were mutant in cdc25 (cdc25-22 arrested in late G2) or cdcl3 (cdc13-117 blocked in midmitosis) was used to demonstrate that the kinase undergoes dephosphorylation on Tyr-15, concomitant with its activation during mitosis. Similarly, in thepatl-114 (ts) mutants, activation of ~ 3 4 ~ ~ at' meiotic ' M phase was correlated with tyrosine dephosphorylation (Figs. 4C and 5). Accumulation of p105wee1 in a high molecular mass complex, which excludes p34cdc2, during the early stages of meiosis in patl-114 (ts) mutants of S. pombe In addition t o reversible phos horylation, the phosphotransferase activity of p34' C2 appears to be modulated by association of the kinase with p13SUC1 and the ~ and their countercyclin B homologue ~ 5 6 ' ~in~S.' pombe parts in other eukaryotes (reviewed in Maller 1990; Nurse 1990; Pelech et al. 1990). Recent studies have indicated that association of p34cdc2with cyclin may facilitate the tyrosyl phosphorylation of p34cdc2 (Parker et al. 1991). A possibility that we sought to address was that ~ 3 4 ~ ~ ~ ' might be maintained in an inactive state prior to meiotic M phase by physical association with ~ 1 0 5 ~In~ preliminary ~'. studies, the adsorption of some of the p105wee1in S. pombe

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DAYA-MAKIN ET AL.

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FIG. 6. Resolution of ~ 1 0 5and ~ ~p34cdcZ ~ ' by density gradient centrifugation. Sedimentation was from left to right. Sucrose density gradient fractions of yeast cells harvested at various times following meiotic induction were probed in Western blots with either antiweel-T peptide antibodies (A-E) or with anti-PSTAIRE antibodies (F-J). The fraction numbers are indicated above the figure. In extracts from mitotic cells, p105we1 was evident throughout the fractions shown (A, 0 h). The tailing observed in later fractions in all cases was probably due to contamination, since the fractions were collected from the top. In extracts from cells committed to meiosis, ~105-I was mainly found in fractions 9-23 corresponding to an intermediate sedimentation rate (A-E), whereas ~ 3 4 remained ~ ~ " in the upper light fractions (F-J).

extracts to p13suc'-agarose beads along with p34cdc2 supported this hypothesis (data not shown). Analysis with sucrose density gradients showed that p l 0 5 ~ occurs ~ ~ ' in high molecular mass complexes during the early stages of meiosis. In extracts from mitotic cells, immunoreactivity to anti-wee1 peptide antibodies was distributed over a broad range of sedimentation rates, consistent with heterogeneity (Fig. 6A). Upon meiotic induction, plO5 was found exclusively in fractions representing intermediate sedimentation rates (Figs. 6B-6E). These results were confirmed by gel filtration on Superose 6, from which the p105wee' complex was released as a broad peak over a range of 150-500 kDa (Fig. 7). The possible association of ~ 1 0 5 " ~with ~ ' other cellular complexes has recently been investigated with yeast mutants

engineered to overexpress this protein (Featherstone and Russell 1991). These studies implied that plO5-' occurs as monomers. However, it has not been resolved whether this represents an artifact due to the overexpression of pl05 wee' (such a system would lead to titration of other components of the complex which are probably present in much lower quantities). Here we have used wild-type cells with respect to the wee1 gene and our data suggest that p l 0 5 ~ is ~ com' plexed to other proteins during meiosis. To determine whether ~ 3 4 ' ~is' ~a component of the p105 complex, the same sucrose density gradient fractions shown in Figs. 6A-6E were also probed with antiPSTAIRE antibodies for ~ 3 4 ' ~ immunoreactivity '~ (Figs. 6F-6J). In those cells that were induced to enter meiosis, peak immunoreactivity with anti-PSTAIRE

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Superose 6 elution volume (mL) FIG. 7. Superose 6 chromatography of p105wee'. Two milligrams of S. pombe protein from GP 48 cells 2 h after shift to the restrictive temperature was resolved by gel filtration chromatography and the p105wee'was detected by ELISA based upon weel-T antibody as described in Materials and methods. Elution positions of the gel filtration marker proteins thyroglobulin (669 kDa), apoferritin (443 kDa), alcohol dehydrogenase(150 kDa), and carbonic anhydrase (29 kDa) are indicated with arrows. The elution behaviour of pl05 from Superose 6 was confirmed by Western blotting analysis (not shown). antibodies (fractions 3-11) did not coincide with peak immunoreactivity with anti-wee1 peptide antibodies (fractions 13- 19). Similarly, when extracts from cells harvested at 2 h postmeiotic induction were chromatographed on Superose 6, the majority of pl~5wee'was resolved from p34cdc2,which fractionated in the range of 30-200 kDa (data not shown). These findings indicated that most of the p34cdc2was not physically associated with pl~5wee1 during meiosis in patl-114 (ts) mutants of S. pombe.

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1992

of pl05"~~'is required for the adequate execution of G2 functions, such as cell growth. Our studies show a decrease following premeiotic replicain the abundance of p105 tion, presumably the meiotic equivalent of mitotic G2. There are some differences in the G2 events between mitosis and meiosis, such as a much shorter G2 phase observed here compared with mitotic G2. Furthermore, meiotic G2 occurs under the conditions of nitrogen starvation and is probably not associated with accumulation in cell mass. Thus, if wee1 functions to ensure that G2 events (such as cell growth) are properly executed prior to cytokinesis, it might be redundant in meiotic G2. Genetic studies using loss of function mutants of p105wee1revealed that these mutants accomplished normal meiosis (Grallert and Sipiczki 1990). Hence, loss of wee1 alone is not sufficient to block meiosis. At semipermissive temperatures, strains of S. pombe carrying the cdc2-33 ts allele were arrested either at START or at ENTRY during vegetative growth, implicating a dual function for cdc2 (Nurse and Bissett 1981; Booher and Beach 1987) in the cell cycle. However, since these mutants did not arrest at meiotic START (Beach et al. 1985), p34cdcZmay not be essential for meiotic replication (which appears to require cdclO (Beach et al. 1985)). These mutants did yield to meiotic arrest but only after the completion of meiosis I, resulting in the formation of asci with two spores (Beach et al. 1985; Hayles et al. 1986; Grallert and Sipiczki 1990). This indicates that unlike meiosis in amphibians and echinoderms, p34cdC2may be required only for the second meiotic division in S. pombe if at all. Our analysis of the stimulation of the kinase activity of ~ 3 4 during ~ ~ ~meiosis ' suggests that it is only required during one of the meiotic divisions in S. pombe. In view of the genetic studies described above, we have inferred that the peak of p34cdc2 kinase activity in our experiments occurred at the second meiotic M phase. Activation of ~ 3 4 was ' ~associated ~ ~ with tyrosine dephosphorylation (Fig. 4C), suggesting a similar mechanism of activation of this kinase in mitosis and meiosis. Indeed ts mutants of S. pombe in the cdc25 gene (a gene encoding a novel ~ 3 4 ' ~phosphatase, '~ originally described during mitotic division in S. pombe) also gave rise to asci containing two spores during meiosis at the semipermissive temperature (Grallert and Sipiczki 1990), again suggesting that it is the second meiotic M phase which has parallels to mitotic M phase.

Discussion In this study, we have demonstrated that thepatl-114 (ts) mutants of S. pombe provide a useful model system for the biochemical analysis of events during the switch from Acknowledgments vegetative growth to meiotic differentiation. We have followed the expression and regulation of ~ 3 4 and ~ ~ ~ ' Drs. Helen Piwnica-Worms and Laura Parker (Tufts University) generously provided baculovirus-expressed plO5 during the entire process of meiosis in S. pombe using this model system. During the early phases of meiosis S. pombe p l 0 5 ~ ~ ~as' ,well as rabbit antisera developed against pl~5wee1. The wee1 deletion mutant of S. pombe (GI and S), there was increase in the steady-state level and tyrosyl phosphorylation of p34cdc2. This coincided with was a gift from Dr. Paul Russell (Scripps Clinic, La Jolla). accumulation of ~ 1 0 5 " ~as~ 'might be anticipated from Dr. Laurent Meijer (Station Biologique, Roscoff) kindly donated the E. coli that expressed p13SUC1.From the mitotic studies. Whether ~ 1 0 5 " ~(or ~ 'p66mik') plays a role in the tyrosine phosphorylation of p34cdc2during this stage Bimedical Research Centre, we gratefully acknowledge Dr. Ian Clark-Lewis, Mr. Philip Owen, and Mr. Peter remains to be established. It has previously been shown that wee1 - mutants appear Borowski for the synthesis and purification of peptides, and to execute mitotic M phase with a much contracted G2 Mr. John Babcook for his aid in the preparation of antiperiod and exhibit a "wee" phenotype (Russell and Nurse peptide antibodies. We also thank Dr. I. Clark-Lewis and 1987a), and cdc2 alleles insensitive to the inhibitory effects Dr. Gerald R. Smith (Fred Hutchinson Cancer Research of weel' have a short G2 and are also phenotypically Centre, Seattle) for their useful discussions and their criti"wee." These results suggest that during mitosis, the activity ques of this manuscript. S.L.P. is the recipient of a Scholar-

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ship from the Medical Research Council of Canada (MRCC). This study was supported by M R C C operating grant t o S.L.P., a n d a United States Public Health Service Grant (no. G M 32194) t o Dr. G. Smith. Arion, D., Meijer, L., Brizuela, L., and Beach, D. 1988. cdc2 is a component of the M phase-specific histone H1 kinase: evidence for identity with MPF. Cell, 55: 371-378. Beach, D., Rodgers, L., and Gould, J. 1985. Rani' controls the transition from mitotic division to meiosis in fission yeast. Curr. Genet. 10: 297-31 1. Booher, R., and Beach, D. 1987. Interactions between cdcl3 and cdc2 in the control of mitosis in fission yeast: dissociation of the G1 and G2 roles of the cdc2' protein kinase. EMBO J. 6: 3441-3447. Booher, R.N., Alfa, C.E., Hyams, J.S., and Beach, D.H. 1989. The fission yeast cdc2/cdcl3/sucl protein kinase: regulation of kinase activity and nuclear localization. Cell, 58: 485-497. Brizuela, L., Draetta, G., and Beach, D. 1987. p13mc' acts in the fission yeast cell division cycle as a component of p34cdc2protein kinase. EMBO J. 6: 3507-3514. Draetta, G., Luca, F., Westendorf, J., Brizuela, L., Ruderman, J., and Beach, D. 1989. cdc2 protein kinase is complexed with both cyclin A and B: evidence for proteolytic inactivation of MPF. Cell, 56: 829-838. Ducommun, B., Draetta, G., Young, P., and Beach, D. 1990. Fission yeast cdc25 is a cell-cycle regulated protein. Biochem. Biophys. Res. Commun. 167: 301-309. Dunphy, W.G., and Kumagai, A. 1991. The cdc25 protein contains an intrinsic phosphatase activity. Cell, 67: 189-196. Dunphy, W.G., Brizuela, L., Beach, D., and Newport, J. 1988. The Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of meiosis. Cell, 54: 423-431. Fantes, P. 1979. Epistatic gene interactions in the control of division in fission yeast. Nature (London), 279: 428-430. Fantes, P.A. 1981. Isolation of cell size mutants of a fission yeast by a new selective method: characterization of mutants and implications for division control mechanisms. J. Bacterial. 146: 746-754. Fantes, P.A., and Nurse, P. 1978. Control of the timing of cell division in fission yeast: cell size mutants reveal a second control pathway. Exp. Cell Res. 115: 317-329. Featherstone, C., and Russell, P. 1991. Fission yeast p107 mitotic inhibitor is a tyrosine/serine kinase. Nature (London), 349: 808-81 1. Feilotter, H., Nurse, P., and Young, P. 1990. Genetic and molecular analysis of cdr-l/nim-1 in Schizosaccharomyces pombe. Genetics, 127: 309-3 18. Gautier, J., Norbury, C., Lohka, M., Nurse, P., and Maller, J. 1988. Purified maturation promoting factor contains the product of a Xenopus homolog of the fission yeast cell cycle control gene cdc2'. Cell, 54: 433-439. Gautier, J., Solomon, M.J., Booher, R.N., Bazan, J.F., and Kirschner, M.W. 1991. cdc25 is a specific tyrosine phosphatase that directly activates p34cdc2.Cell, 67: 197-21 1. Gould, K.L., and Nurse, P. 1989. Tyrosine phosphorylation of the fission yeast cdc2+ protein kinase regulates entry into mitosis. Nature (London). 342: 39-45. Gould, K.L., Moreno, S., Tonks, N.K., and Nurse, P. 1990. Complementation of the mitotic activator p8~cdc25 by a human protein-tyrosine phosphatase. Nature (London), 250: 1573-1576. Gould, K.L., Moreno. S., Owen, D.J., Sazer, S., and Nurse, P. 1991. Phosphorylation at Thr167 is required for Schizosaccharomycespombe p34cdc2function. EMBO J. 10: 3297-3309. Grallert, B., and Sipiczki, M. 1990. Dissociation of the meiotic and mitotic roles of the fission yeast cdc2 gene. Mol. Gen. Genet. 222: 473-475. +

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