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Purified maturation-promoting factor contains the product of a Xenopus homolog of the fission yeast cell cycle control gene cdc2+
Cell, Vol. 54, 433-439,
July 29, 1988,
Copyright
0 1988 by Cell Press
Purified Maturation-Promoting Factor Contains the Product of a Xenopus Homolog of the Fission Yeast Cell Cycle Control Gene c&2+ Jean Gautier: Chris Norbury,t Manfred Lohka,” * Paul Nurse,t and James Maker’ “Department of Pharmacology University of Colorado School of Medicine Denver, Colorado 80262 f ICRF Cell Cycle Control Laboratory Microbiology Unit Department of Biochemistry University of Oxford Oxford OX13QU, England
Summary In the fission yeast S. pombe, the M, = 34 kd product of the cdc2+ gene (~34~d~2) is a protein kinase that controls entry into mitosis. In Xenopus oocytes and other cells, maturation-promoting factor (MPF) appears in late 62 phase and is able to cause entry into mitosis. Purified MPF consists of two major proteins of M r = 32 kd and 45 kd and expresses protein kinase activity. We report here that antibodies to S. pombe ~34~~~~ are able to immunoblot and immunoprecipitate the ~32 kd component of MPF from Xenopus eggs. The M, = 32 kd and 45 kd proteins exist as a complex that expresses protein kinase activity. These findings indicate that a Xenopus ~34~“~ homolog is present in purified MPF and suggest that p34Cdc2 is a component of the control mechanism initiating mitosis generally in eukaryotic cells. Introduction One of the most fundamental problems in cell biology concerns the control of the eukaryotic cell cycle. Changes in the regulation of the cycle are clearly evident in neoplastic cells and during early development, implicating cell cycle control as a major target for understanding the basis of both cancer and morphogenesis. In most eukaryotes, the cell cycle consists of several phases, in the simplest case a mitotic (M) phase in which chromosomes are distributed equally to daughter cells and an S phase in which DNA is replicated. While cells in early embryos such as Xenopus exhibit only M and S (Graham and Morgan, 1966), most cells exhibit two additional phases involving a Gl phase between mitosis and S phase and a G2 phase between S phase and mitosis. Two general control points have been identified in the cell cycle, one in Gl acting over entry into S phase and one in late G2 acting over entry into mitosis. These controls have been described in a wide range of cells, from yeast to humans, suggesting that they are fundamental features of the cell cycle. Analysis of the G2/M control point has been primarily * Present University
address: Department of Cellular and Structural Biology, of Colorado School of Medicine, Denver, Colorado 80282.
carried out with cells of the fission yeast Schizosaccharomyces pombe (Nurse, 1985 for review) and of the frog Xenopus laevis, although mammalian cells can also be arrested in G2 under certain experimental conditions (Gelfant, 1962; Pedersen and Gelfant, 1970; Nose and Katsuta, 1975; Stambrook and Velez, 1976; Melchers and Lernhardt, 1985). In fission yeast, a regulatory network of genes has been identified as controlling entry into mitosis. A protein kinase designated ~34~~~ encoded by the cd&‘+ gene is controlled by a number of positive (cdc25+, niml+), and negative (weel+) regulatory factors (Nurse, 19751985; Nurse and Thuriaux, 1980; Russell and Nurse, 1986; Simanis and Nurse, 1986). These factors act together to effect an orderly transition from G2 into mitosis and are thought to respond to various signals important for mitotic initiation, such as those generated by changes in cell size and growth rate (Fantes and Nurse, 1977) and the completion of S phase. The niml+ and weel+ genes encode proteins with consensus sequences for protein kinases (Russell and Nurse, 1987a, 1987b), implicating protein phosphorylation in the activation of the ~34~~~~ protein kinase for the initiation of mitosis. The c&2+ gene is structurally similar and functionally interchangeable with the cell cycle gene CDC28 in the distantly related budding yeast Saccharomyces cerevisiae (Beach et al., 1982). In addition, a human homolog of ccfczC has been isolated by complementation, which is able to substitute for the functions encoded by c&2+ in yeast, suggesting that elements of the mitotic control are conserved in all eukaryotic cells (Lee and Nurse, 1987). This idea is further supported by the ability of antibody to ~34~~~’ to recognize homologs in S. cerevisiae and in HeLa cells (Draetta et al., 1987). In frogs, information about events at the G2/M transition point has come largely from studies with oocytes (Mailer, 1985, for review). Xenopus oocytes are arrested physiologically in late G2, and reentry into the cell cycle occurs in response to a variety of mitogenic agents, notably progesterone, insulin, or IGF,. Several hours later, shortly before germinal vesicle (nuclear) breakdown, an activity appears in the cytoplasm that is believed to be directly responsible for triggering the initiation of mitotic events. This activity, known as maturation-promoting factor (MPF), was described as a factor present in mature oocyte cytoplasm that upon microinjection could cause G2-arrested oocytes to enter meiosis even in the absence of protein synthesis (Masui and Markert, 1971; Smith and Ecker, 1971). Subsequent work has revealed that an activity with identical properties appeared during M phase in a variety of oocytes, in somatic cells entering mitosis (Kishimoto et al., 1982,1984; Sunkara et al., 1979) and in budding yeast (Tachibana et al., 1987). These results support the concept that MPF is a fundamental and universal regulator of entry into M phase. Other evidence strongly suggested that the biochemical basis of MPF action, like p34cdc2, was protein phosphorylation. When MPF appears in cells or is injected into
Cell 434
oocytes of various species, there is an immediate increase in total protein phosphorylation (Maker et al., 1977; Doree et al., 1983) which includes the appearance of new phosphoproteins (Maker and Smith, 1985). Moreover, MPF activity cannot be extracted from cells in the absence of several putative phosphatase inhibitors, notably j3-glycerophosphate, indicating that MPF might contain a protein kinase or an activator of a protein kinase. Purification of MPF has provided support for this hypothesis. Initially, little progress was made in purifying MPF using the Xenopus oocyte microinjection assay, partly because activity could only be detected in concentrated fractions (Wu and Gerhart, 1980). Extracts from unfertilized Rana eggs with MPF activity were shown to cause sperm nuclei to form chromosomes in vitro (Lohka and Masui, 1984), and this system was extended to Xenopus eggs by Lohka and Maller (1985) who showed that the addition of partially purified MPF to nuclei resulted in nuclear envelope breakdown, chromosome condensation, and spindle formation. In addition, the phosphoproteins observed to accompany MPF action in vivo were also found to become phosphorylated in the cell-free system after MPF addition (Lohka et al., 1987). The development of an MPF-dependent cell-free system for early mitotic events formed the basis for a new assay to assist in MPF purification. By monitoring breakdown of pronuclei assembled in vitro, we recently purified MPF 3500-fold from unfertilized Xenopus eggs (Lohka et al., 1988). Purified MPF was active not only in causing nuclear breakdown in the cell-free system but also in causing germinal vesicle breakdown when microinjected into cycloheximide-treated Xenopus oocytes. The purified preparation consisted largely of two polypeptides of apparent molecular weight 32,000 and 45,000. The purified MPF expressed a protein kinase activity that phosphorylated the M, = 45,000 protein as well as histone Hl, casein, and phosphatase inhibitor 1 (Lohka et al., 1988). Because ~34~~~ and MPF both appear to act as fundamental regulators of entry into mitosis and are found in a wide range of eukaryotic cells, it is important to establish whether these two activities are related. This is of particular interest given that purified MPF contains a protein of similar molecular weight to ~34~“~. We report here that antibodies against ~34~~~~ both immunoblot and immunoprecipita:e the ~32 kd component of MPF, indicating that these two proteins are very closely related and probably similar in function. Results cdc2 Antibody lmmunoblots an ~34 kd Protein in Purified MPF Previous studies have shown that the predicted amino acid sequence of the human CDC2 homolog exhibits 83% overall identity with that of S. pombe cdczC (Lee and Nurse, 1987) and that certain regions in ~34~~~ are perfectly conserved between yeast and humans. One such region, of 18 amino acid residues (EGVPSTAIREISLLKE), was used to generate a rabbit polyclonal antibody (Lee and Nurse, 1987). lmmunoblotting of extracts from a range of eukaryotic cells has shown that this antibody (called
PSTAIR) specifically recognizes a single protein of M, of approximately 34,000. This antibody also detects a single protein in Xenopus egg extracts. lmmunoblotting of crude ammonium sulfate fractions of egg extract revealed a single reactive component migrating with an M, = 34,000 (Figure la). In the 00%34% ammonium sulfate fraction, which specifically precipitates active MPF (Wu and Gerhart, 1980; Lohka et al., 1988), ~34~~~ migrated slightly more slowly (Figure la, lane 1) than in the 340/o-100% fraction (Figure la, lane 2). No reaction occurred with normal rabbit serum, and the reaction was fully blocked by preincubation of the antibody with the synthetic peptide (Figure la, lane 3). More importantly, the antibody also specifically recognized the M, = 34,000 protein in a highly purified MPF fraction eluted from a Mono S column (Figure lb, lane 2). This protein was found to comigrate with p34c*2 encoded by the human homolog of c&2’+ (Figure lb, lane 3). The fractions that retained full MPF activity across the final Mono S column also contained the M, = 34,000 protein (Figure lc, lanes 11 and 12). The same fractions also contained the protein found in purified MPF and previously described to be of M, = 32,000 (Lohka et al., 1988). Fraction 13 contained partial MPF activity and also contained the M, = 34,000 protein. cdc2 Antibody lmmunoprecipitates a 34 kd Protein in Purified MPF To obtain further evidence that ~34~~~~ is related to the M, = 32,000 protein in MPF, immunoprecipitation of purified MPF was investigated. Previous studies have shown that incubation of purified MPF with [Y-~~P]ATP results in phosphorylation of the M, = 45,000 protein in the final purified Mono S preparation (Lohka et al., 1988). Upon long autoradiographic exposures, labeling of the M, = 32,000 component is also evident, and this labeling is more efficient at earlier stages of the purification in the peak fraction from the TSK 3000 SW column. Peak fractions of MPF from both the TSK 3000 SW and Mono S columns were incubated with [y-ssP]ATP and then subjected to immunoprecipitation with the PSTAIR antibody. As shown in Figures 2a and 2b, a labeled protein of M, =: 34,000 was precipitated from the MPF peak fractions of both columns. Hydrolysis of the labeled protein revealed that the majority of phosphorylation was on threonine residues (data not shown). No immunoprecipitation was seen with normal rabbit serum nor if peptide was preincubated with the antibody prior to additon of the peak fractions (Figures 2a and 2b). Because the PSTAIR antibody efficiently immunoprecipitates material from the Mono S column, which is only labeled inefficiently, it is possible that the antibody preferentially precipitates the phosphorylated form of the Xenopus protein. A second antibody raised against the carboxy-terminal region of human ~34~“~ also immunoprecipitated the M, = 34,000 protein (data not shown). These results, together with the immunoblotting data, indicate that purified MPF contains a protein closely related to ~34~“~. The immunoprecipitates were also found to contain the M, = 45,000 protein component of purified MPF (Figures 2a and 2b). This protein was not immunoprecipitated with
p34m2 435
Is Present
A
12
in MPF
Figure 1. lmmunoblotting body to ~34~~
3
-67
443
>430
420
~34~~~~ Peptide Accelerates MPF Activity These results suggest that MPF contains a Xenopus homolog of ~34~~~. To examine further the relationship between ~34~~~~ and MPF activity, we attempted to immunodeplete ~34~~~ from purified MPF in order to test the ability of the depleted activity to induce nuclear envelope breakdown and chromosome condensation in vitro. Unfortunately, the cross reactive antibodies were unable to completely immunodeplete ~34~~~ from purified MPF (data not shown), which prevented us from further pursu-
with
Anti-
(A) Ammonium sulfate fractions of high speed supernatants from unfertilized eggs. High speed supernatants from unfertilized Xenopus eggs were prepared as described previously (Lohka et al., 1966) and subjected to O%-34% ammonium sulfate precipitation. Aliquotsof the precipitated and solubfe fractiins were resolved on SDS polyacrylamide gels and immunoblotted with PSTAIR antibody as described in Experimental Procedures. An autoradiograph of the blot is shown. The numbers to the right indicate the positions to which molecular weight standards migrated. Lane 1, O%-34% ammonium sulfate precipitated fraction; lane 2, O%34% ammonium sulfate soluble fraction; lane 3, O%-34% ammonium sulfate precipitated fraction with antibody previously incubated with the 16mer synthetic peptide corresponding to the conserved sequence of ~34~~~. (B) MPF purified through Mono S chromatography. lmmunoblotting was performed as described above. Lane 1. total E. coli protein (negative control); lane 2, a peak MPF fraction from a Mono S column; lane 3, total protein from ICRF23 human fibroblasts. (C) MPF-positive and negative Mono S fractions. lmmunoblotting was performed as described in (A). Each lane contains an aliquot of protein from the Mono S fraction series described by Lohka and Mailer (1966); the number of each fraction is indicated, together with the presence (+) or absence (-) of full MPF activity, as judged by the ability to induce nuclear envelope breakdown (NEED) in vitro. ‘+” indicates induction of 100% NEBD within 60 min; lower levels of activity were detected in fractions to either side of fractions 11 and 12.
497
normal rabbit serum if peptide was preincubated with the antibody prior to addition of the peak fractions. This suggests that the two proteins exist as a complex, resulting in their coimmunoprecipitation. The immunoprecipitate also had protein kinase activity, shown when the peak TSK 3000 SW fraction was immunoprecipitated with the PSTAIR antibody, and the immune precipitate incubated with histone Hl and [T-32P]ATf? Phosphorylation of histone Hi was evident, which could be completely blocked by preincubation of the antibody with peptide (Figure 2~). This result is consistent with the observations that both MPF and ~34~~~ have protein kinase activity (Simanis and Nurse, 1986; Lohka et al., 1988).
of MPF
ing this experimental approach. We next tested the effects on MPF activity of the peptide corresponding to the 16 amino acid region of ~34~~~~ perfectly conserved from yeasts to humans. Because this region of the protein is so conserved, it seems likely that it interacts with another component in the mitotic control regulatory network that might influence ~34~~~~ function. Conceivably, addition of the peptide could compete with ~34~~~~ for interaction with another component and thus influence entry into mitosis. Addition of the peptide alone did not cause any change in nuclear morphology. However, when the peptide was added together with MPF to pronuclei assembled in vitro in Xenopus egg extracts, there was a marked acceleration of the rate at which MPF caused nuclear breakdown and chromosome condensation (Figure 3). In the presence of peptide, MPF-induced nuclear envelope breakdown and chromosome condensation occurred 30 min earlier relative to controls utilizing buffer or another peptide with no homology to ~34~~~. Discussion The results in this paper provide evidence that a Xenopus protein homologous to S. pombe ~34~~~~ is a component
Cell 436
67,
Figure
2. lmmunoprecipitation
of MPF by Antipeptide
Antibody
(A) MPF purified through TSK chromatography. A peak MPF fraction from a TSK 3000 SW column was incubated with [Y~‘P]ATP immunoprecipitated with antibody that had (lane 1) or had not (lane 2) been preincubated with the 16mer peptide corresponding to the conserved region of ~34~~’ as described under Experimental Procedures, and subjected to SDS gel electrophoresis. An autoradiograph of the gel is shown. (B) MPF purified through Mono S chromatography. A peak MPF fraction from a Mono S column was incubated with [Y-~*P]ATP immunoprecipitated as described in (A) with antibody that had (lane 1) or had not (lane 2) been preincubated with the 16mer peptide. (C) Hl kinase activity of immunoprecipitated MPF. MPF from a peak fraction of TSK 3000 SW columns was immunoprecipitated, and the immunoprecipitate was used to phosphorylate histone Hi as described in Experimental Procedures. An autoradiograph of the reaction products is shown after SDS gel electrophoresis. The antibody either had (lane 2) or had not (lane 1) been preincubated with the 16mer peptide.
Time
after
MPF
AdI
Figure 3. Acceleration of Nuclear Envelope some Condensation by a ~34~~~~ Peptide
tion,
min
Breakdown
and Chromo-
Pronuclei were assembled in vitro in Xenopus egg extracts and incubated for 20 min with 40 ng of the synthetic peptide corresponding to a conserved domain in ~34~~~. A second addition of 40 ng of peptide was then made along with 2 U of MPF activity and the time course of nuclear events monitored as described in Experimental Procedures. Open bars, buffer addition; cross-hatched bars, control peptide addition; filled bars, ~34~~’ peptide. Data are expressed as the extent of nuclear response in arbitrary units, where 0 represents less than 20% pronuclear breakdown (Lohkaet al., 1966); 1 represents approximately 50% nuclear breakdown and chromosome condensation, and 2 represents induction of these events in 90%-100% of the pronuclei.
of MPF. An antibody directed against ~34~~2 detects the 32-34 kd protein component of highly purified MPF (Figure 1). This antibody is very specific, having been raised against a domain perfectly conserved from the yeasts to humans, and detects only a single protein of approximately 34 kd in crude extracts of a range of eukaryotic cells. Thus, two independent lines of work have led to the identification of a single protein species as a key component in the initiation of mitosis. The biochemical approach in Xenopus has led to the purification of a homolog of p34Ck2 as a component of MPF, and the genetic approach in fission yeast has led to the cloning of the cdczC gene encoding ~34~~’ as an element in the regulatory gene network controlling mitotic initiation. Purified fractions of MPF contain not only a M, =: 32 kd protein but also a M, = 45 kd protein, suggesting that both may be needed for MPF activity. Our immunoprecipitation results demonstrate that these two proteins can exist as a complex. The immunoprecipitated ~34~~~ can also be labeled with [Y-~‘P]ATP in vitro. Incubation of the TSK 3000 and Mono S fractions with [y-s2P]ATP leads to the formation of labeled p34dC2 and labeled 45 kd protein in immunoprecipitates (Figure 2). It is likely that this labeling of p34CN2 is inefficient, as the majority of the ~34~~~ present before immunoprecipitation is not highly labeled compared with that observed for the 45 kd protein (Lohka et al., 1988). The ability of MPF immunoprecipitated with
p34Ce2 437
Is Present
in MPF
cdc2 antibody to express Hl histone kinase activity provides evidence this activity is due to the ~34~~~ component since ~34~~C2 from S. pombe has also been found to phosphorylate Hl histones (Moreno and Nurse, unpublished data). The phosphorylation of Hl histone may be physiologically significant since phosphorylation of this protein is known to increase in mitotic cells (Ajiro et al., 1983; Mueller et al., 1985). The 45 kd protein may be of interest for two reasons. Firstly, niml+, a putative protein kinase activator of p34C”z functions, is predicted to encode a protein of M, = 45 kd (Russell and Nurse, 1987b). A Xenopus homolog of this protein could be necessary to activate ~34~~~ in the purified MPF complex. Secondly, a potential substrate has been identified for ~34~o~** (the budding yeast homolog of p34CdC2) of approximate M, = 40 kd (Mendenhall et al., 1987). In purified Mono S preparations of MPF, the 45 kd protein only becomes phosphorylated in fractions that also contain ~34~~~~ (Lohka et al., 1988), and it is therefore possible that the 45 kd protein is a substrate. Given that the amount of ~34~~~ in yeast does not change during the cell cycle, it is possible that the association of Xenopus ~34~~~~ with the M, = 45 kd protein regulates MPF activity. lmmunoblotting data (Figure 1) show that the cdc2 antibody recognized ~34~~~ in ammonium sulphate fractions with MPF activity as well as those without detectable activity. It was notable that the ~34~~~ in active MPF fractions migrated on a l-dimensional gel more slowly than in nonactive fractions, suggesting that a modified, possibly phosphorylated, form of ~34~~~~ might be enriched in active MPF fractions. Consistent with this idea, MPF is known to be stored in an inactive form in Xenopus oocytes (Reynhout and Smith, 1974; Cyert and Kirchner, 1988). In addition, it should be remembered that the cdczC gene in fission yeast is required at the Gl/S boundary of the cell cycle as well as at G2/M (Nurse, 1985). It is possible that different modified forms of ~34~~~ are required at both points in the cell cycle and that only the form arising in G2 complexed with the 45 kd component has MPF activity. The importance of ~34~~~ in MPF activity is supported by the finding that a peptide corresponding to a highly conserved domain in ~34~~~~ can accelerate MPF action, strengthening our view that p34@* and a component of MPF are related functionally. It is conceivable that the peptide could have a direct activating effect on an Mphase regulatory component. However, MPF inactivating systems are known to occur in vivo (Gerhart et al., 1984) and in vitro (Cyert and Kirschner, 1988), and it is also possible that the peptide could compete for an inactivating system, allowing MPF to work at an effective higher dose. In the cell-free system, higher doses of MPF are able to cause more rapid nuclear envelope breakdown and chromosome condensation. The fact that the effect is largely on the time course of nuclear breakdown could also reflect the rapid destruction of the peptide in egg extracts, as we have observed previously with other synthetic peptides. In any case, the ability of the peptide to affect MPF action supports the hypothesis that cdc2 activity is involved in MPF action.
The linking together of Xenopus and fission yeast mitotic controls will enable both biochemical and genetic approaches to be combined in the study of initiation of mitosis. Proteins associated with MPF and identified as potential substrates in Xenopus can be compared with gene products thought to interact with p34** function in yeast. These studies should be useful in establishing how the G2 to mitosis transition is controlled and how the early events of mitosis are implemented. We are encouraged in our belief that the basic mechanism of mitotic initiation is conserved in all eukaryotic cells and that ~34~“~ is a key component of the control exerted by MPF. Experimental
Procedures
MPF Purification MPF was purified from unfertilized scribed (Lohka et al., 1988).
Xenopus
eggs
as previously
de-
Antibody Preparation and lmmunochemical Aeeaye Antisera and affinity-purified antibody were prepared as previously described (Lee and Nurse, 1987). For immunoblotting, proteins were transferred from 10% polyacrylamide gels (Laemmli, 1970) containing 10% glycerol to nitrocellulose fillers using a semidry blotting system (Nova-Blot, LKB) at 0.8 mA/cm* for 55 min in 50 mM Tris, 39 mM glytine. 0.0375% SDS, and 20% methanol. The nitrocellulose filter was soaked in blocking solution (10% dry milk in phosphate buffered saline [PBS] for 1 hr at room temperature under constant agitation, and then the filter was incubated overnight at 4OC with the affinity-purified antibody in PBS containing 10% dry milk and 0.3% Tween 20. The filter was washed several times at room temperature with PBS containing 0.3% Tween 20 and then incubated with the same solution containing [1251]-protein A (lo6 dpmlml) for 1 hr at room temperature. The filter was washed several times in PBSmween 20 with the last rinse being in PBS containing 0.3% Tween 20, 1% Briton X100, and 0.05% SDS for IO min. Control experiments were performed using affinity-purified antibody previously incubated for 1 hr at 4OC with the 18mer peptide (1 nglpl of affinity-purified serum). lmmunoprecipitations using anti-cdc2 antibody were performed using either [r-32P]ATP labeled samples or unlabeled samples for subsequent HI kinase assays. In a typical experiment, 50 ul of MPF from either the TSK or the Mono S chromatography step was incubated in 25 pl of protein A-Sepharose (Pharmacia) for 1 hr at 4% under constant agitation. The protein A-resin had previously been equilibrated in RIPA buffer (20 mM Tris [pH 7.41, 5 mM EDTA. 100 mM NaCI, 1% Triton X-100) containing 300 mM polymethylsulfonyl fluoride (PMSF), 5 @ml of leupeptin, 1 mM Na pyrophosphate, 1 mM Na vanadate, 10 mM Na fluoride, 1 mM EGTA, and 1 mM p-glycerophosphate. The mixture was centrifuged at 15,000 x g for 1 min, and the supernatant was used for immunoprecipitation. In case of =P-labeling, prior to immunoprecipitation, the sample was incubated for 15 min at 3oOC with 1 x l@ cpm of [@2P]ATR at a final ATP concentration of 0.1 mM. The reaction was stopped by the addition of Edit to a final concentration of 10 mM. In all cases, the sample was then incubated overnight at 4OC under moderate agitation with 3 pl of affinity-purified serum, after which 25 ~1 of protein A-Sepharose was added and the sample incubated for 1 hr. Following centrifugation and removal of the supernatant, the protein A-resin was washed quickly two times with RIPA buffer and a third time for 1 hr, then washed twice in RIPA containing 1 M NaCI, and twice in RIPA containing 100 mM NaCI. After removing the supernatant, the resin was processed either for elactrophoresis (in the case of 32P-labeled extract) or for Hl kinase assay. Protein Kinase Aeeays The histone Hl assay was performed in a final volume of 40 pl containing 20 mM HEPES, 30 mM 8-mercaptoethanol, 0.1 mglml of BSA, 10 mM MgCI, 0.5 mg/ml of rat thymus histone Hl (gift of Dr. T. A. Langan, Department of Pharmacology), and 0.1 mM ATP containing 1 x lo8 cpm of [Y-~“P]ATP. Reactions were incubated at WC for 15 min and stopped by the addition of Vi vol of 5x electrophoresis sample buffer.
Cdl 438
Control experiments were done using affinity purified had been previously incubated with 1 ng of the 16mer of antibody for 1 hr at 4OC.
Aeeay
of Peptlde
Effect
antibody that peptide per ~1
on MPF Activity
We thank Karen Eckart for secretarial assistance. This work was supported by grants to J. L. M. from the National Institutes of Health and the American Cancer Society and by ICRF funding to R N. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “ad~rtisemenf” in accordance with 18 USC. Section 1734 solely to indicate this fact. June
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Extracts able to cause pronuclear formation in vitro were prepared as previously described (Lohka and Mailer, 1965) and incubated with demembranated sperm nuclei for 1 hr at 16oC. At this stage, 1 ~1 of the peptide was added to 12.5 pl of the pronuclear suspension, and the mixture was incubated for 20 min at 16V. Then another 1 ui aliquot of the peptide was added with 25 PI of MPF containing 2 U of activity, and the progression of nuclear envelope breakdown was monitored by taking 5 ~1 aliquots of the mixture every 15 min, mixing with an equal volume of a 5 pg/ml of 4,6-Diamidino-2-phenylindole (DAPI) solution, and observing nuclear morphology by phase contrast and fluorescence microscopy. Peptide concentrations ranging from 4-140 ng/$ were assayed. The most effective peptide concentration was found to be 30-40 ng/pl. The control for MPF activity was performed by adding MPF to the extract with either buffer or a control peptide (AETAAAAKFLRAAA) whose sequence was unrelated to ~34~~. Data are expressed as the extent of nuclear response in arbitrary units, where 0 represents less than 20% pronuclear breakdown (Lohka et al., 1966); 1 represents approximately 50% nuclear breakdown and chromosome condensation, and 2 represents induction of these events in 900/b-100% of the pronuclei.
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Gerhart, J., Wu, M., and Kirschner, M. W. (1964). Cell cycle dynamics of an M-phase cytoplasmic factor in Xenopus laevis oocytes and eggs. J. Cell Biol. 98, 1247-1255.
Reynhout, J. K., and Smith, L. D. (1974). Studies of the appearance and nature of a maturation-inducing factor in the cytoplasm of amphibian oocytes exposed to progesterone. Dev. Biol. 25, 232-247.
Graham, C. F.. and Morgan, R. W. (1966). Changes in the cell cycle during early amphibian development. Dev. Biol. 14, 349-361.
Russell, P., and Nurse, I? (1986). cdc25+ functions as an Inducer the mitotic control of fission yeast. Cell 45, 145-153.
Kishimoto, T., Kuriyama, R., Kondo, H., and Kanatani. H. (1982). Generality of the action of various maturation-promoting factors. Exp. Cell Res. 137; 121-126.
Russell, P., and Nurse, weef+, a gene encoding
Kishimoto. T., Yamazaki, K., Kato, Y., Koide, S. S., and Kanatani, H. (1964). Induction of starfish oocyte maturation by maturation-
in
P. (1967a). Negative regulation of mitosis by a protein kinase homolog. Cell 49, 559-567.
Russell, P., and Nurse, I? (1967b). The mitotic inducer niml+ in a regulatory network of protein kinase homologs controlling of mitosis. Cell 49, 569-576.
functions initiation
p34cdc2 Is Present 439
in MPF
Simanis. V., and Nurse, P (;966). The cell cycle control gene cd& of fission yeast encodes a protein kinase potentially regulated by phosphorylation. Cell 45, 261-266. Smith, L. D., and Ecker, R. E. (1971). The interaction Rana pipiens oocytes in the induction of maturation. 233-247.
of steroids with Dev. Biol. 25,
Stambrook, f?, and Velez, C. (1976). Reversible arrest of Chinese hamster V79 cells in G2 by dibutyryl cyclic AMP Exp. Cell Res. 99, 57-62. Sunkara, 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, 2799-2602. Tachibana. K., Yanagashima, N., and Kishimoto, T. (1967). Preliminary characterization of maturation-promoting factor from yeast Saccharomyces cerevisiae. J. Cell Sci. 88, 273-261. Wu, M., and Gerhart, J. C. (1960). Partial purification and characterization of the maturation-promoting factor from eggs of Xenopus laevis. Dev. Biol. 79, 465-477.
July 29, 1988,
Copyright
0 1988 by Cell Press
Purified Maturation-Promoting Factor Contains the Product of a Xenopus Homolog of the Fission Yeast Cell Cycle Control Gene c&2+ Jean Gautier: Chris Norbury,t Manfred Lohka,” * Paul Nurse,t and James Maker’ “Department of Pharmacology University of Colorado School of Medicine Denver, Colorado 80262 f ICRF Cell Cycle Control Laboratory Microbiology Unit Department of Biochemistry University of Oxford Oxford OX13QU, England
Summary In the fission yeast S. pombe, the M, = 34 kd product of the cdc2+ gene (~34~d~2) is a protein kinase that controls entry into mitosis. In Xenopus oocytes and other cells, maturation-promoting factor (MPF) appears in late 62 phase and is able to cause entry into mitosis. Purified MPF consists of two major proteins of M r = 32 kd and 45 kd and expresses protein kinase activity. We report here that antibodies to S. pombe ~34~~~~ are able to immunoblot and immunoprecipitate the ~32 kd component of MPF from Xenopus eggs. The M, = 32 kd and 45 kd proteins exist as a complex that expresses protein kinase activity. These findings indicate that a Xenopus ~34~“~ homolog is present in purified MPF and suggest that p34Cdc2 is a component of the control mechanism initiating mitosis generally in eukaryotic cells. Introduction One of the most fundamental problems in cell biology concerns the control of the eukaryotic cell cycle. Changes in the regulation of the cycle are clearly evident in neoplastic cells and during early development, implicating cell cycle control as a major target for understanding the basis of both cancer and morphogenesis. In most eukaryotes, the cell cycle consists of several phases, in the simplest case a mitotic (M) phase in which chromosomes are distributed equally to daughter cells and an S phase in which DNA is replicated. While cells in early embryos such as Xenopus exhibit only M and S (Graham and Morgan, 1966), most cells exhibit two additional phases involving a Gl phase between mitosis and S phase and a G2 phase between S phase and mitosis. Two general control points have been identified in the cell cycle, one in Gl acting over entry into S phase and one in late G2 acting over entry into mitosis. These controls have been described in a wide range of cells, from yeast to humans, suggesting that they are fundamental features of the cell cycle. Analysis of the G2/M control point has been primarily * Present University
address: Department of Cellular and Structural Biology, of Colorado School of Medicine, Denver, Colorado 80282.
carried out with cells of the fission yeast Schizosaccharomyces pombe (Nurse, 1985 for review) and of the frog Xenopus laevis, although mammalian cells can also be arrested in G2 under certain experimental conditions (Gelfant, 1962; Pedersen and Gelfant, 1970; Nose and Katsuta, 1975; Stambrook and Velez, 1976; Melchers and Lernhardt, 1985). In fission yeast, a regulatory network of genes has been identified as controlling entry into mitosis. A protein kinase designated ~34~~~ encoded by the cd&‘+ gene is controlled by a number of positive (cdc25+, niml+), and negative (weel+) regulatory factors (Nurse, 19751985; Nurse and Thuriaux, 1980; Russell and Nurse, 1986; Simanis and Nurse, 1986). These factors act together to effect an orderly transition from G2 into mitosis and are thought to respond to various signals important for mitotic initiation, such as those generated by changes in cell size and growth rate (Fantes and Nurse, 1977) and the completion of S phase. The niml+ and weel+ genes encode proteins with consensus sequences for protein kinases (Russell and Nurse, 1987a, 1987b), implicating protein phosphorylation in the activation of the ~34~~~~ protein kinase for the initiation of mitosis. The c&2+ gene is structurally similar and functionally interchangeable with the cell cycle gene CDC28 in the distantly related budding yeast Saccharomyces cerevisiae (Beach et al., 1982). In addition, a human homolog of ccfczC has been isolated by complementation, which is able to substitute for the functions encoded by c&2+ in yeast, suggesting that elements of the mitotic control are conserved in all eukaryotic cells (Lee and Nurse, 1987). This idea is further supported by the ability of antibody to ~34~~~’ to recognize homologs in S. cerevisiae and in HeLa cells (Draetta et al., 1987). In frogs, information about events at the G2/M transition point has come largely from studies with oocytes (Mailer, 1985, for review). Xenopus oocytes are arrested physiologically in late G2, and reentry into the cell cycle occurs in response to a variety of mitogenic agents, notably progesterone, insulin, or IGF,. Several hours later, shortly before germinal vesicle (nuclear) breakdown, an activity appears in the cytoplasm that is believed to be directly responsible for triggering the initiation of mitotic events. This activity, known as maturation-promoting factor (MPF), was described as a factor present in mature oocyte cytoplasm that upon microinjection could cause G2-arrested oocytes to enter meiosis even in the absence of protein synthesis (Masui and Markert, 1971; Smith and Ecker, 1971). Subsequent work has revealed that an activity with identical properties appeared during M phase in a variety of oocytes, in somatic cells entering mitosis (Kishimoto et al., 1982,1984; Sunkara et al., 1979) and in budding yeast (Tachibana et al., 1987). These results support the concept that MPF is a fundamental and universal regulator of entry into M phase. Other evidence strongly suggested that the biochemical basis of MPF action, like p34cdc2, was protein phosphorylation. When MPF appears in cells or is injected into
Cell 434
oocytes of various species, there is an immediate increase in total protein phosphorylation (Maker et al., 1977; Doree et al., 1983) which includes the appearance of new phosphoproteins (Maker and Smith, 1985). Moreover, MPF activity cannot be extracted from cells in the absence of several putative phosphatase inhibitors, notably j3-glycerophosphate, indicating that MPF might contain a protein kinase or an activator of a protein kinase. Purification of MPF has provided support for this hypothesis. Initially, little progress was made in purifying MPF using the Xenopus oocyte microinjection assay, partly because activity could only be detected in concentrated fractions (Wu and Gerhart, 1980). Extracts from unfertilized Rana eggs with MPF activity were shown to cause sperm nuclei to form chromosomes in vitro (Lohka and Masui, 1984), and this system was extended to Xenopus eggs by Lohka and Maller (1985) who showed that the addition of partially purified MPF to nuclei resulted in nuclear envelope breakdown, chromosome condensation, and spindle formation. In addition, the phosphoproteins observed to accompany MPF action in vivo were also found to become phosphorylated in the cell-free system after MPF addition (Lohka et al., 1987). The development of an MPF-dependent cell-free system for early mitotic events formed the basis for a new assay to assist in MPF purification. By monitoring breakdown of pronuclei assembled in vitro, we recently purified MPF 3500-fold from unfertilized Xenopus eggs (Lohka et al., 1988). Purified MPF was active not only in causing nuclear breakdown in the cell-free system but also in causing germinal vesicle breakdown when microinjected into cycloheximide-treated Xenopus oocytes. The purified preparation consisted largely of two polypeptides of apparent molecular weight 32,000 and 45,000. The purified MPF expressed a protein kinase activity that phosphorylated the M, = 45,000 protein as well as histone Hl, casein, and phosphatase inhibitor 1 (Lohka et al., 1988). Because ~34~~~ and MPF both appear to act as fundamental regulators of entry into mitosis and are found in a wide range of eukaryotic cells, it is important to establish whether these two activities are related. This is of particular interest given that purified MPF contains a protein of similar molecular weight to ~34~“~. We report here that antibodies against ~34~~~~ both immunoblot and immunoprecipita:e the ~32 kd component of MPF, indicating that these two proteins are very closely related and probably similar in function. Results cdc2 Antibody lmmunoblots an ~34 kd Protein in Purified MPF Previous studies have shown that the predicted amino acid sequence of the human CDC2 homolog exhibits 83% overall identity with that of S. pombe cdczC (Lee and Nurse, 1987) and that certain regions in ~34~~~ are perfectly conserved between yeast and humans. One such region, of 18 amino acid residues (EGVPSTAIREISLLKE), was used to generate a rabbit polyclonal antibody (Lee and Nurse, 1987). lmmunoblotting of extracts from a range of eukaryotic cells has shown that this antibody (called
PSTAIR) specifically recognizes a single protein of M, of approximately 34,000. This antibody also detects a single protein in Xenopus egg extracts. lmmunoblotting of crude ammonium sulfate fractions of egg extract revealed a single reactive component migrating with an M, = 34,000 (Figure la). In the 00%34% ammonium sulfate fraction, which specifically precipitates active MPF (Wu and Gerhart, 1980; Lohka et al., 1988), ~34~~~ migrated slightly more slowly (Figure la, lane 1) than in the 340/o-100% fraction (Figure la, lane 2). No reaction occurred with normal rabbit serum, and the reaction was fully blocked by preincubation of the antibody with the synthetic peptide (Figure la, lane 3). More importantly, the antibody also specifically recognized the M, = 34,000 protein in a highly purified MPF fraction eluted from a Mono S column (Figure lb, lane 2). This protein was found to comigrate with p34c*2 encoded by the human homolog of c&2’+ (Figure lb, lane 3). The fractions that retained full MPF activity across the final Mono S column also contained the M, = 34,000 protein (Figure lc, lanes 11 and 12). The same fractions also contained the protein found in purified MPF and previously described to be of M, = 32,000 (Lohka et al., 1988). Fraction 13 contained partial MPF activity and also contained the M, = 34,000 protein. cdc2 Antibody lmmunoprecipitates a 34 kd Protein in Purified MPF To obtain further evidence that ~34~~~~ is related to the M, = 32,000 protein in MPF, immunoprecipitation of purified MPF was investigated. Previous studies have shown that incubation of purified MPF with [Y-~~P]ATP results in phosphorylation of the M, = 45,000 protein in the final purified Mono S preparation (Lohka et al., 1988). Upon long autoradiographic exposures, labeling of the M, = 32,000 component is also evident, and this labeling is more efficient at earlier stages of the purification in the peak fraction from the TSK 3000 SW column. Peak fractions of MPF from both the TSK 3000 SW and Mono S columns were incubated with [y-ssP]ATP and then subjected to immunoprecipitation with the PSTAIR antibody. As shown in Figures 2a and 2b, a labeled protein of M, =: 34,000 was precipitated from the MPF peak fractions of both columns. Hydrolysis of the labeled protein revealed that the majority of phosphorylation was on threonine residues (data not shown). No immunoprecipitation was seen with normal rabbit serum nor if peptide was preincubated with the antibody prior to additon of the peak fractions (Figures 2a and 2b). Because the PSTAIR antibody efficiently immunoprecipitates material from the Mono S column, which is only labeled inefficiently, it is possible that the antibody preferentially precipitates the phosphorylated form of the Xenopus protein. A second antibody raised against the carboxy-terminal region of human ~34~“~ also immunoprecipitated the M, = 34,000 protein (data not shown). These results, together with the immunoblotting data, indicate that purified MPF contains a protein closely related to ~34~“~. The immunoprecipitates were also found to contain the M, = 45,000 protein component of purified MPF (Figures 2a and 2b). This protein was not immunoprecipitated with
p34m2 435
Is Present
A
12
in MPF
Figure 1. lmmunoblotting body to ~34~~
3
-67
443
>430
420
~34~~~~ Peptide Accelerates MPF Activity These results suggest that MPF contains a Xenopus homolog of ~34~~~. To examine further the relationship between ~34~~~~ and MPF activity, we attempted to immunodeplete ~34~~~ from purified MPF in order to test the ability of the depleted activity to induce nuclear envelope breakdown and chromosome condensation in vitro. Unfortunately, the cross reactive antibodies were unable to completely immunodeplete ~34~~~ from purified MPF (data not shown), which prevented us from further pursu-
with
Anti-
(A) Ammonium sulfate fractions of high speed supernatants from unfertilized eggs. High speed supernatants from unfertilized Xenopus eggs were prepared as described previously (Lohka et al., 1966) and subjected to O%-34% ammonium sulfate precipitation. Aliquotsof the precipitated and solubfe fractiins were resolved on SDS polyacrylamide gels and immunoblotted with PSTAIR antibody as described in Experimental Procedures. An autoradiograph of the blot is shown. The numbers to the right indicate the positions to which molecular weight standards migrated. Lane 1, O%-34% ammonium sulfate precipitated fraction; lane 2, O%34% ammonium sulfate soluble fraction; lane 3, O%-34% ammonium sulfate precipitated fraction with antibody previously incubated with the 16mer synthetic peptide corresponding to the conserved sequence of ~34~~~. (B) MPF purified through Mono S chromatography. lmmunoblotting was performed as described above. Lane 1. total E. coli protein (negative control); lane 2, a peak MPF fraction from a Mono S column; lane 3, total protein from ICRF23 human fibroblasts. (C) MPF-positive and negative Mono S fractions. lmmunoblotting was performed as described in (A). Each lane contains an aliquot of protein from the Mono S fraction series described by Lohka and Mailer (1966); the number of each fraction is indicated, together with the presence (+) or absence (-) of full MPF activity, as judged by the ability to induce nuclear envelope breakdown (NEED) in vitro. ‘+” indicates induction of 100% NEBD within 60 min; lower levels of activity were detected in fractions to either side of fractions 11 and 12.
497
normal rabbit serum if peptide was preincubated with the antibody prior to addition of the peak fractions. This suggests that the two proteins exist as a complex, resulting in their coimmunoprecipitation. The immunoprecipitate also had protein kinase activity, shown when the peak TSK 3000 SW fraction was immunoprecipitated with the PSTAIR antibody, and the immune precipitate incubated with histone Hl and [T-32P]ATf? Phosphorylation of histone Hi was evident, which could be completely blocked by preincubation of the antibody with peptide (Figure 2~). This result is consistent with the observations that both MPF and ~34~~~ have protein kinase activity (Simanis and Nurse, 1986; Lohka et al., 1988).
of MPF
ing this experimental approach. We next tested the effects on MPF activity of the peptide corresponding to the 16 amino acid region of ~34~~~~ perfectly conserved from yeasts to humans. Because this region of the protein is so conserved, it seems likely that it interacts with another component in the mitotic control regulatory network that might influence ~34~~~~ function. Conceivably, addition of the peptide could compete with ~34~~~~ for interaction with another component and thus influence entry into mitosis. Addition of the peptide alone did not cause any change in nuclear morphology. However, when the peptide was added together with MPF to pronuclei assembled in vitro in Xenopus egg extracts, there was a marked acceleration of the rate at which MPF caused nuclear breakdown and chromosome condensation (Figure 3). In the presence of peptide, MPF-induced nuclear envelope breakdown and chromosome condensation occurred 30 min earlier relative to controls utilizing buffer or another peptide with no homology to ~34~~~. Discussion The results in this paper provide evidence that a Xenopus protein homologous to S. pombe ~34~~~~ is a component
Cell 436
67,
Figure
2. lmmunoprecipitation
of MPF by Antipeptide
Antibody
(A) MPF purified through TSK chromatography. A peak MPF fraction from a TSK 3000 SW column was incubated with [Y~‘P]ATP immunoprecipitated with antibody that had (lane 1) or had not (lane 2) been preincubated with the 16mer peptide corresponding to the conserved region of ~34~~’ as described under Experimental Procedures, and subjected to SDS gel electrophoresis. An autoradiograph of the gel is shown. (B) MPF purified through Mono S chromatography. A peak MPF fraction from a Mono S column was incubated with [Y-~*P]ATP immunoprecipitated as described in (A) with antibody that had (lane 1) or had not (lane 2) been preincubated with the 16mer peptide. (C) Hl kinase activity of immunoprecipitated MPF. MPF from a peak fraction of TSK 3000 SW columns was immunoprecipitated, and the immunoprecipitate was used to phosphorylate histone Hi as described in Experimental Procedures. An autoradiograph of the reaction products is shown after SDS gel electrophoresis. The antibody either had (lane 2) or had not (lane 1) been preincubated with the 16mer peptide.
Time
after
MPF
AdI
Figure 3. Acceleration of Nuclear Envelope some Condensation by a ~34~~~~ Peptide
tion,
min
Breakdown
and Chromo-
Pronuclei were assembled in vitro in Xenopus egg extracts and incubated for 20 min with 40 ng of the synthetic peptide corresponding to a conserved domain in ~34~~~. A second addition of 40 ng of peptide was then made along with 2 U of MPF activity and the time course of nuclear events monitored as described in Experimental Procedures. Open bars, buffer addition; cross-hatched bars, control peptide addition; filled bars, ~34~~’ peptide. Data are expressed as the extent of nuclear response in arbitrary units, where 0 represents less than 20% pronuclear breakdown (Lohkaet al., 1966); 1 represents approximately 50% nuclear breakdown and chromosome condensation, and 2 represents induction of these events in 90%-100% of the pronuclei.
of MPF. An antibody directed against ~34~~2 detects the 32-34 kd protein component of highly purified MPF (Figure 1). This antibody is very specific, having been raised against a domain perfectly conserved from the yeasts to humans, and detects only a single protein of approximately 34 kd in crude extracts of a range of eukaryotic cells. Thus, two independent lines of work have led to the identification of a single protein species as a key component in the initiation of mitosis. The biochemical approach in Xenopus has led to the purification of a homolog of p34Ck2 as a component of MPF, and the genetic approach in fission yeast has led to the cloning of the cdczC gene encoding ~34~~’ as an element in the regulatory gene network controlling mitotic initiation. Purified fractions of MPF contain not only a M, =: 32 kd protein but also a M, = 45 kd protein, suggesting that both may be needed for MPF activity. Our immunoprecipitation results demonstrate that these two proteins can exist as a complex. The immunoprecipitated ~34~~~ can also be labeled with [Y-~‘P]ATP in vitro. Incubation of the TSK 3000 and Mono S fractions with [y-s2P]ATP leads to the formation of labeled p34dC2 and labeled 45 kd protein in immunoprecipitates (Figure 2). It is likely that this labeling of p34CN2 is inefficient, as the majority of the ~34~~~ present before immunoprecipitation is not highly labeled compared with that observed for the 45 kd protein (Lohka et al., 1988). The ability of MPF immunoprecipitated with
p34Ce2 437
Is Present
in MPF
cdc2 antibody to express Hl histone kinase activity provides evidence this activity is due to the ~34~~~ component since ~34~~C2 from S. pombe has also been found to phosphorylate Hl histones (Moreno and Nurse, unpublished data). The phosphorylation of Hl histone may be physiologically significant since phosphorylation of this protein is known to increase in mitotic cells (Ajiro et al., 1983; Mueller et al., 1985). The 45 kd protein may be of interest for two reasons. Firstly, niml+, a putative protein kinase activator of p34C”z functions, is predicted to encode a protein of M, = 45 kd (Russell and Nurse, 1987b). A Xenopus homolog of this protein could be necessary to activate ~34~~~ in the purified MPF complex. Secondly, a potential substrate has been identified for ~34~o~** (the budding yeast homolog of p34CdC2) of approximate M, = 40 kd (Mendenhall et al., 1987). In purified Mono S preparations of MPF, the 45 kd protein only becomes phosphorylated in fractions that also contain ~34~~~~ (Lohka et al., 1988), and it is therefore possible that the 45 kd protein is a substrate. Given that the amount of ~34~~~ in yeast does not change during the cell cycle, it is possible that the association of Xenopus ~34~~~~ with the M, = 45 kd protein regulates MPF activity. lmmunoblotting data (Figure 1) show that the cdc2 antibody recognized ~34~~~ in ammonium sulphate fractions with MPF activity as well as those without detectable activity. It was notable that the ~34~~~ in active MPF fractions migrated on a l-dimensional gel more slowly than in nonactive fractions, suggesting that a modified, possibly phosphorylated, form of ~34~~~~ might be enriched in active MPF fractions. Consistent with this idea, MPF is known to be stored in an inactive form in Xenopus oocytes (Reynhout and Smith, 1974; Cyert and Kirchner, 1988). In addition, it should be remembered that the cdczC gene in fission yeast is required at the Gl/S boundary of the cell cycle as well as at G2/M (Nurse, 1985). It is possible that different modified forms of ~34~~~ are required at both points in the cell cycle and that only the form arising in G2 complexed with the 45 kd component has MPF activity. The importance of ~34~~~ in MPF activity is supported by the finding that a peptide corresponding to a highly conserved domain in ~34~~~~ can accelerate MPF action, strengthening our view that p34@* and a component of MPF are related functionally. It is conceivable that the peptide could have a direct activating effect on an Mphase regulatory component. However, MPF inactivating systems are known to occur in vivo (Gerhart et al., 1984) and in vitro (Cyert and Kirschner, 1988), and it is also possible that the peptide could compete for an inactivating system, allowing MPF to work at an effective higher dose. In the cell-free system, higher doses of MPF are able to cause more rapid nuclear envelope breakdown and chromosome condensation. The fact that the effect is largely on the time course of nuclear breakdown could also reflect the rapid destruction of the peptide in egg extracts, as we have observed previously with other synthetic peptides. In any case, the ability of the peptide to affect MPF action supports the hypothesis that cdc2 activity is involved in MPF action.
The linking together of Xenopus and fission yeast mitotic controls will enable both biochemical and genetic approaches to be combined in the study of initiation of mitosis. Proteins associated with MPF and identified as potential substrates in Xenopus can be compared with gene products thought to interact with p34** function in yeast. These studies should be useful in establishing how the G2 to mitosis transition is controlled and how the early events of mitosis are implemented. We are encouraged in our belief that the basic mechanism of mitotic initiation is conserved in all eukaryotic cells and that ~34~“~ is a key component of the control exerted by MPF. Experimental
Procedures
MPF Purification MPF was purified from unfertilized scribed (Lohka et al., 1988).
Xenopus
eggs
as previously
de-
Antibody Preparation and lmmunochemical Aeeaye Antisera and affinity-purified antibody were prepared as previously described (Lee and Nurse, 1987). For immunoblotting, proteins were transferred from 10% polyacrylamide gels (Laemmli, 1970) containing 10% glycerol to nitrocellulose fillers using a semidry blotting system (Nova-Blot, LKB) at 0.8 mA/cm* for 55 min in 50 mM Tris, 39 mM glytine. 0.0375% SDS, and 20% methanol. The nitrocellulose filter was soaked in blocking solution (10% dry milk in phosphate buffered saline [PBS] for 1 hr at room temperature under constant agitation, and then the filter was incubated overnight at 4OC with the affinity-purified antibody in PBS containing 10% dry milk and 0.3% Tween 20. The filter was washed several times at room temperature with PBS containing 0.3% Tween 20 and then incubated with the same solution containing [1251]-protein A (lo6 dpmlml) for 1 hr at room temperature. The filter was washed several times in PBSmween 20 with the last rinse being in PBS containing 0.3% Tween 20, 1% Briton X100, and 0.05% SDS for IO min. Control experiments were performed using affinity-purified antibody previously incubated for 1 hr at 4OC with the 18mer peptide (1 nglpl of affinity-purified serum). lmmunoprecipitations using anti-cdc2 antibody were performed using either [r-32P]ATP labeled samples or unlabeled samples for subsequent HI kinase assays. In a typical experiment, 50 ul of MPF from either the TSK or the Mono S chromatography step was incubated in 25 pl of protein A-Sepharose (Pharmacia) for 1 hr at 4% under constant agitation. The protein A-resin had previously been equilibrated in RIPA buffer (20 mM Tris [pH 7.41, 5 mM EDTA. 100 mM NaCI, 1% Triton X-100) containing 300 mM polymethylsulfonyl fluoride (PMSF), 5 @ml of leupeptin, 1 mM Na pyrophosphate, 1 mM Na vanadate, 10 mM Na fluoride, 1 mM EGTA, and 1 mM p-glycerophosphate. The mixture was centrifuged at 15,000 x g for 1 min, and the supernatant was used for immunoprecipitation. In case of =P-labeling, prior to immunoprecipitation, the sample was incubated for 15 min at 3oOC with 1 x l@ cpm of [@2P]ATR at a final ATP concentration of 0.1 mM. The reaction was stopped by the addition of Edit to a final concentration of 10 mM. In all cases, the sample was then incubated overnight at 4OC under moderate agitation with 3 pl of affinity-purified serum, after which 25 ~1 of protein A-Sepharose was added and the sample incubated for 1 hr. Following centrifugation and removal of the supernatant, the protein A-resin was washed quickly two times with RIPA buffer and a third time for 1 hr, then washed twice in RIPA containing 1 M NaCI, and twice in RIPA containing 100 mM NaCI. After removing the supernatant, the resin was processed either for elactrophoresis (in the case of 32P-labeled extract) or for Hl kinase assay. Protein Kinase Aeeays The histone Hl assay was performed in a final volume of 40 pl containing 20 mM HEPES, 30 mM 8-mercaptoethanol, 0.1 mglml of BSA, 10 mM MgCI, 0.5 mg/ml of rat thymus histone Hl (gift of Dr. T. A. Langan, Department of Pharmacology), and 0.1 mM ATP containing 1 x lo8 cpm of [Y-~“P]ATP. Reactions were incubated at WC for 15 min and stopped by the addition of Vi vol of 5x electrophoresis sample buffer.
Cdl 438
Control experiments were done using affinity purified had been previously incubated with 1 ng of the 16mer of antibody for 1 hr at 4OC.
Aeeay
of Peptlde
Effect
antibody that peptide per ~1
on MPF Activity
We thank Karen Eckart for secretarial assistance. This work was supported by grants to J. L. M. from the National Institutes of Health and the American Cancer Society and by ICRF funding to R N. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “ad~rtisemenf” in accordance with 18 USC. Section 1734 solely to indicate this fact. June
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Extracts able to cause pronuclear formation in vitro were prepared as previously described (Lohka and Mailer, 1965) and incubated with demembranated sperm nuclei for 1 hr at 16oC. At this stage, 1 ~1 of the peptide was added to 12.5 pl of the pronuclear suspension, and the mixture was incubated for 20 min at 16V. Then another 1 ui aliquot of the peptide was added with 25 PI of MPF containing 2 U of activity, and the progression of nuclear envelope breakdown was monitored by taking 5 ~1 aliquots of the mixture every 15 min, mixing with an equal volume of a 5 pg/ml of 4,6-Diamidino-2-phenylindole (DAPI) solution, and observing nuclear morphology by phase contrast and fluorescence microscopy. Peptide concentrations ranging from 4-140 ng/$ were assayed. The most effective peptide concentration was found to be 30-40 ng/pl. The control for MPF activity was performed by adding MPF to the extract with either buffer or a control peptide (AETAAAAKFLRAAA) whose sequence was unrelated to ~34~~. Data are expressed as the extent of nuclear response in arbitrary units, where 0 represents less than 20% pronuclear breakdown (Lohka et al., 1966); 1 represents approximately 50% nuclear breakdown and chromosome condensation, and 2 represents induction of these events in 900/b-100% of the pronuclei.
Received
promoting 293-295.
M. W. (1968).
Regulation
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