INTRODUCTION

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Eukaryotic cell cycle progression requires the sequential differential regulation of cyclin-dependent protein kinases (CDKs). In general, CDK activity can be regulated at four posttranslational levels (reviewed in reference 37): the binding of a positive regulator (a cyclin), the binding of a negative regulator (a CDK inhibitor), activating phosphorylation at threonine 169 (Saccharomyces cerevisiae) or at homologous sites in other organisms (e.g., T161 in Schizosaccharomyces pombe), or inhibiting phosphorylation at tyrosine 19 (S. cerevisiae) or at homologous sites (e.g., Y15 in S. pombe).

By regulating the state of phosphorylation of tyrosine 15 of the CDK Cdc2p, organisms such as S. pombe, Xenopus laevis, and humans can effectively coordinate the timing of the entry into mitosis with the correct completion of DNA synthesis (5, 20, 39, 53) or with an assessment of the integrity of the genome (5, 20, 38). The phosphorylation of Y15 in normally growing cells also has the effect of maintaining Cdc2 kinase activity at low levels so as to prevent the ectopic initiation of mitotic events (9). Wee1 kinase and its homologs phosphorylate Y15; Cdc25 phosphatase and its homologs remove this phosphate. In S. pombe, mutations in the genes encoding Wee1p and Cdc25p can elicit severe mitotic defects (44, 45). When DNA replication is blocked or DNA is damaged, a checkpoint signal causes an enhancement of Wee1 function and an inhibition of Cdc25 activity, thereby causing cells to arrest at G₂/M and preventing premature entry into mitosis. In S. cerevisiae, the phosphorylation (and dephosphorylation) of tyrosine 19 on its major CDK, Cdc28p, is not required for mitotic timing, nor is it required for DNA replication or DNA damage checkpoint-mediated cell cycle arrest (1, 55). Rather, regulating Y19 phosphorylation of Cdc28 in S. cerevisiae appears to be related to checkpoint mechanisms associated with monitoring the morphological integrity of the cell, be it the actin cytoskeleton (28, 47) or the septin ring of the developing bud neck (3). When the actin cytoskeleton is disrupted either by drug treatment or the effect of conditional mutations, Swe1p, the S. cerevisiae Wee1p homolog, is stabilized, thereby conferring a mitotic delay. When septin function is compromised, Hsl1, Gin4, and Kcc4 kinases redundantly mediate a Swe1-dependent delay of entry into mitosis. It is not known whether these two pathways share components or not.

Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase whose function has been implicated in a variety of cellular functions including DNA replication, cell cycle progression, RNA transcription, RNA splicing, and translation (60). With regard to its cell cycle function, PP2A has been identified as a negative regulator of entry into mitosis. For example, a factor purified from X. laevis egg extracts that prevents the activation of Cdc2 kinase activity was shown to be a form of PP2A (25). It was also reported that PP2A activity, as inferred from treatment with okadaic acid, a relatively specific PP2A inhibitor, was required to maintain high levels of Xe-Wee1p (the Xenopus Wee1p homolog) activity during DNA replication in X. laevis egg extracts (53). More recently, in this same system, it was shown that okadaic acid increased the rate of degradation of Xe-Wee1p and prematurely induced mitosis in the presence of a DNA replication checkpoint; inactivating the Skp1-Cdc53/Cul1-F-box protein (SCF)-ubiquitinating complex prevented this ectopic mitotic entry, most likely through the stabilization of Xe-Wee1p (32). In S. pombe, loss of one of the two PP2A catalytic subunits causes a wee1-like phenotype and is synthetically lethal with certain mutant alleles of wee1⁺. This same deletion partially suppressed the growth defect of cdc25 mutants, supporting the notion that PP2A acts as a positive regulator of Wee1 and/or as a negative regulator of Cdc25 (23). By contrast, in S. cerevisiae, PP2A appears to be a positive regulator of entry into mitosis. In a situation in which PP2A catalytic activity was depleted from the cell, Clb2/Cdc28 kinase activity became severely reduced and cells arrested as large budded 2N cells at G₂/M (28). This raises two questions: what role, if any, does PP2A play in S. cerevisiae with regard to the regulation of phosphorylation of Y19 of Cdc28p, and, if required for this regulation, through what mechanisms does it achieve that control?

PP2A is thought to function primarily as a heterotrimeric complex composed of a catalytic subunit (C) and two others (A and B) serving regulatory functions (reviewed in references 34, 56, 58, and 60). The A subunit in S. cerevisiae is encoded by TPD3 (57). It serves as a structural platform to which one of the two B-regulatory subunits, Rts1p (46) or Cdc55p (16), and one of the catalytic subunits, Pph21p or Pph22p (40, 54), bind. The deletion of either PPH21 or PPH22 elicits no mutant phenotype, but the loss of Pph21p, Pph22p, and Pph3p (a protein sequence-related to Pph21p and Pph22p) causes abnormal bud shape, abnormal actin cytoskeleton, a G₂/M delay, and a weakened cell wall (14, 28). Deletion of either RTS1 or CDC55 produces cells with completely different mutant phenotypes. rts1-null cells are temperature sensitive for growth at 37°C and are defective in their ability to respond to a number of physiological stresses (13, 46, 47). By contrast, cdc55-null cells grow slowly at low temperature and exhibit an abnormal budding morphology (16). They are also defective in establishing a spindle damage checkpoint, losing viability rapidly when exposed to normally nontoxic doses of nocodazole or benomyl (35, 59). rts1-null and cdc55-null cells share no apparent overlap of phenotypic deficiencies. The double-null rts1 cdc55 mutant exhibits a combination of the two single-null phenotypes (46).

At low temperatures, cdc55-null cells exhibit an elongation of buds and a failure of cytokinesis and cell separation (16). The abnormal morphology is abolished in these cells if they also express a CDC28 gene in which Y19 is changed to F19, thereby rendering the inhibitory phosphorylation site on Cdc28p nonphosphorylatable (59). When measured directly, the phosphorylation level on tyrosine 19 of Cdc28 in cdc55-null cells is higher in cycling and nocodazole-arrested cells than in similarly treated wild-type cells (35). These results suggested a probable role for PP2A in regulating Cdc28 tyrosine phosphorylation.

We have addressed the question of what causes the elevated phosphorylation levels of Cdc28 Y19 in cdc55-null cells. More specifically, we wanted to know if PP2A activity is required for the regulation of either Swe1 kinase activity or Mih1 (the S. cerevisiae Cdc25 homolog) phosphatase activity and, if so, how losing Cdc55p disrupts this regulation. What we have found is that the loss of Cdc55 affects the normal regulation of Swe1p turnover, causing cells to accumulate abnormally high levels of this protein at mitosis. Furthermore, this ectopic accumulation and resultant excess Swe1 kinase activity are likely caused by a gain of a formerly repressed PP2A catalytic function. Our data also show that, as in other eukaryotes, PP2A function is required in S. cerevisiae for regulating the inhibitory phosphorylation of its CDK.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Plasmids and strains. All strains used were derivatives of W303. The wild-type and cdc55-null (cdc55::TRP1) strains have been previously described (46). A cdc55-null swe1-null strain was generated by transformation of the cdc55-null strain by one-step gene disruption (42) using a BamHI-HindIII-digested swe1::LEU2 knockout construct (6). W303 strains null for PPH21 and PPH22 (DEY132-1C [pph21::HIS3] and DEY10-2B [pph22::URA3]) were obtained from D. Evans. Strains null for CDC55 and one or both of the genes encoding the PP2A catalytic subunits were generated by crosses, tetrad dissection, and phenotypic screening to identify the appropriate genotypic segregants. W303 strains carrying chromosomally integrated copies of CDC28:HA (ADR508) or Y19F-CDC28:HA (ADR640) were obtained from A. Murray. These genes were introduced into the appropriate deletion strains by standard crosses. A W303 strain (PS701) containing a MIH1 knockout (mih1::LEU2) was obtained from P. Sorger. This strain and the cdc55-null strain were individually crossed to ADR508 to obtain mih1CDC28:HA and cdc55CDC28:HA segregants, respectively.

mat MIH1:MYC₁₂

cdc55 pph21 pph22 SWE1

cdc55::TRP1 SWE1

pph21::HIS3 pph22::URA3

swe1::LEU2

cdc55::TRP1 swe1::LEU2

Cell growth conditions. Standard yeast protocols and media were used (41). To arrest cell growth, hydroxyurea was added to culture media to a final concentration of 0.2 M; nocodazole was added to a final concentration of 20 µg/ml. In all cases, cells were microscopically examined (either by differential interference-contrast [DIC] for cell morphology or 4',6'-diamidino-2-phenylindole [DAPI] staining/fluorescence for nuclear morphology) throughout all experiments to ensure that cell cycle arrest had occurred and that such arrest was maintained. The exact conditions used for specific experiments are given in the appropriate figure legends.

DAPI staining and microscopy. Staining cells with DAPI to determine the state of arrest of cells was carried out by the method of Honigberg and Esposito (17). Cells growing in liquid cultures were ethanol fixed directly in growth medium. Cells growing on solid medium were scraped off, washed once in water, and then fixed. For quantitative measurements, 200 to 300 cells were counted for each time point. Visualization of cells by DIC microscopy was carried out on a Nikon Eclipse TE300 microscope. Visualization of cells using DAPI or green fluorescent protein (GFP) fluorescence was done on an Olympus BX60 microscope. For quantitation purposes, images were captured using an Optronics cooled charge-coupled device camera.

Northern analyses. Cells to be analyzed were centrifuged and stored as frozen pellets at 80°C. Total RNAs were prepared using a hot-phenol extraction method (24). RNAs were denatured, separated on 1% agarose gels, transferred to filters, and hybridized with a radioactive probe as previously described (47). An Swe1 mRNA probe was generated by labeling a 0.6-kb HindIII-XbaI SWE1 ORF fragment with [-³²P]ATP, using a random labeling kit from Life Technologies.

Protein isolation, electrophoresis, and Western analysis. The direct extraction of total proteins by solubilizing cells in 1.8 M NaOH-5% beta-mercaptoethanol (-ME), the separation of proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and procedures used for Western analysis have been previously described (46, 47). The antibody against phospho-Tyr15-CDC2 (New England Biolabs) was used following the instructions of the supplier. The mouse monoclonal antibodies 12CA5 and 9E10 were used to detect HA epitope-tagged proteins and MYC epitope-tagged proteins, respectively. Cdc28p and Pho85p were detected using the mouse PSTAIR monoclonal antibody (Sigma). The final visualization of proteins was done using alkaline phosphatase-conjugated second antibodies as previously described (47) or by using horseradish peroxidase (HRP)-conjugated second antibodies and enhanced chemiluminescence (ECL; Amersham).

Mih1p in vitro phosphatase activity assay. Growing CDC55 and cdc55 strains expressing an MIH1-MYC gene were arrested in nocodazole for 3 h. As negative controls, CDC55 and cdc55 cells expressing a normal MIH1 gene were treated in the same way. Such cells were harvested and frozen at 80°C. Subsequently thawed cells were broken by glass beads at 4°C in an immunoprecipitation (IP) buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM EGTA, 0.1% NP-40, 2.7 mM KCl) supplemented with a protease inhibitor cocktail (Boehringer Mannheim) and 1 mM phenylmethylsulfonate fluoride (PMSF). Following cell breakage, the lysates of the four samples were centrifuged at high speed at 4°C for 5 min (all subsequent steps were carried out at 4°C unless specified otherwise). The lysates were transferred to clean centrifuge tubes, and to each was added 20 µl of a 1:1 slurry of protein A beads equilibrated with IP buffer. The tubes were gently agitated for 20 min, after which they were centrifuged at high speed for 5 min. Each of the cleared lysates (ca. 0.5 ml) was removed and transferred to a clean centrifuge tube, and 4 µl of 9E10 anti-myc mouse monoclonal antibody (1 mg/ml; Zymed) was added. These tubes were then gently agitated for 40 min, after which 100 µl of a 1:1 protein A bead-IP buffer slurry was added and agitation was continued for an additional 45 min. The protein A beads were collected by centrifugation at 4,000 × g for 5 s. The supernate was then removed, and the beads were washed three times with 1 ml of IP solution and then twice with 1 ml of phosphatase assay buffer (50 mM Tris-Cl [pH 8.0], 50 mM NaCl, 1 mM EDTA). Following the second wash, one-tenth of the beads were collected by centrifugation and boiled in 20 µl of 2× SDS-PAGE loading buffer. These samples were used to detect the amount and modification of Mih1p in each immunoprecipitate before the phosphatase assay. The myc-tagged Mih1p from the CDC55- and cdc55-null cells adsorbed to the protein A beads served as the source of phosphatase to be assayed in vitro.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

cdc55-null cells remain arrested in mitosis when treated with nocodazole. It was shown (35) that cdc55-null cells have an increased level of phosphorylation of Cdc28p tyrosine 19 (Y19) when compared with wild-type controls. The extent of phosphorylation at Y19 is controlled by two counteracting enzymes: Swe1p (a kinase) and Mih1p (a phosphatase). Thus, assuming that no other previously unrecognized enzymes are involved, either higher Swe1 kinase activity or lower Mih1 phosphatase activity in cdc55-null cells accounts for the increased Cdc28p Y19-phosphorylation level. Our goal was to establish which alternative applied for cdc55-null cells.

View larger version (55K): [in this window] [in a new window]   FIG. 1.   Nocodazole-treated cdc55 cells remain arrested in mitosis. To determine whether nocodazole-treated cdc55-null cells maintain a mitotic arrest, we examined the metabolism of Pds1p and Cln2p in these cells and compared it to that seen in wild-type controls. To examine Pds1p initially, isogenic wild-type (CDC55) and cdc55 strains, each transformed with a plasmid carrying an HA-tagged PDS1 gene behind a GAL1 promoter (see Materials and Methods), were grown in raffinose medium to early log phase. Nocodazole was then added to the culture medium to 20 µg/ml to arrest cell growth, and 3 h later, galactose was added to induce the accumulation of HA-tagged Pds1p. After 2 h, cells were collected by centrifugation, washed once, and then, to repress further PDS1 transcription, resuspended in dextrose medium containing nocodazole to maintain mitotic arrest. Cell samples were collected immediately and at 0.5-h intervals for 2 h after PDS1 turnoff. Total cell proteins (see Materials and Methods) were separated by SDS-PAGE, transferred to nitrocellulose filters, and immunoblotted using 12CA5 anti-HA antibody to detect Pds1p (A). The lower half of the gel not used for the protein transfer was stained with Coomassie blue to show the relative protein loading in each lane. Endogenous Pds1p levels were determined in CDC55 and cdc55-null strains at various times during an extended exposure to nocodazole or after a release from a 3-h nocodazole arrest (B). Wild-type and cdc55-null cells, each expressing an HA-tagged PDS1 gene (see Materials and Methods), were grown to early log phase in YPD medium. Nocodazole was added to both cultures, and aliquots of cells were collected at 0, 1, 2, and 3 h. After 3 h in nocodazole, the remaining wild-type and cdc55 cultures were split in half. One-half of the cells remained in YPD containing nocodazole. The other cells were collected, washed, and then resuspended in fresh YPD containing no nocodazole to allow cells to enter the cell cycle. Aliquots of cells were collected 20, 40, 60, 80, and 100 min later. Western analyses were carried out to measure Pds1p levels (upper panels), and those portions of the gels not used for protein transfer were stained to show the relative loading in each lane. Lanes 1 through 4, cells in nocodazole at 0, 1, 2, and 3 h; lanes 5 through 9, cells at 20, 40, 60, 80, or 100 min after nocodazole release; lanes 10 through 14, cells in nocodazole at 20, 40, 60, 80, or 100 min after the initial 3-h drug incubation. Cln2p levels were determined in CDC55 and cdc55-null strains at various times during an extended exposure to nocodazole or after release from a 3-h nocodazole arrest (C). Wild-type and cdc55-null cells, each expressing an HA-tagged CLN2 gene (see Materials and Methods), were grown to early log phase in YPD medium. Nocodazole was added to both cultures, and aliquots of cells were collected at 0, 1, 2, and 3 h. After 3 h in nocodazole, the remaining wild-type and cdc55 cultures were split in half. One-half of the cells remained in YPD containing nocodazole. The other cells were collected, washed, and then resuspended in fresh YPD containing no nocodazole to allow cells to reenter the cell cycle. Aliquots of cells were collected 1, 2, and 3 h later. Western blot analyses were carried out to measure Cln2p levels (upper panels), and that portion of the gel not used for protein transfer was stained to show the relative loading in each lane. Lanes 1 through 7, cells in nocodazole at 0, 1, 2, 3, 4, 5, and 6 h; lanes 8 through 11, cells at 0, 1, 2, and 3 h after nocodazole release at 3 h. Note: lanes 4 and 8 contain the same protein samples.

Mih1 phosphatase activity is similar in cdc55-null and wild-type cells. As the hyperphosphorylation of tyrosine 19 of Cdc28p was positively correlated with (and appeared to be directly responsible for) the morphological abnormalities manifested in cdc55-null cells (35, 59), we investigated the possible causes of this state of excess phosphorylation. Swe1 kinase and Mih1 phosphatase are the only reported S. cerevisiae enzymes that either add or remove phosphates from the Y19 residue of Cdc28p. Thus, either higher Swe1 kinase activity or lower Mih1 phosphatase activity in cdc55-null cells must account for the increased phosphorylation state of Cdc28p.

View larger version (69K): [in this window] [in a new window]   FIG. 2.   Mih1p levels in wild-type and cdc55-null cells are similar. To determine Mih1p levels, wild-type (i.e., CDC55) and cdc55-null strains expressing an MIH1 gene tagged with 12 copies of the MYC epitope were constructed (see Materials and Methods). Such cells growing in YPD medium at 30°C were collected as unsynchronized cycling cells (lanes 1 and 2), cells arrested in S phase with 0.2 M HU for 3 h (lanes 3 and 4), or cells arrested in mitosis with 20 µg of nocodazole (noc) per ml for 3 h (lanes 5 and 6). Total cell proteins were extracted as described above, separated by SDS-PAGE, transferred to filters, and immunodecorated using the 9E10 anti-MYC antibody to detect Mih1p (a). The lower half of the gel not used for the protein transfer was stained with Coomassie blue to show the relative protein loading. To determine whether the heterogeneity of Mih1p gel mobility seen in (a) was due to differential phosphorylation of Mih1p, proteins were extracted from HU-arrested wild-type and cdc55 cells, immunoadsorbed to protein A beads, and treated with alkaline phosphatase (see Materials and Methods). Proteins released from the protein A beads were subjected to a Western blot analysis (b) as above. (p), untreated controls; (+p), alkaline phosphatase treated.

View larger version (48K): [in this window] [in a new window]   FIG. 3.   Measuring Mih1 phosphatase activity in wild-type and cdc55-null cells. To test the ability of the anti-phospho-Tyr15-Cdc2 antibody to detect S. cerevisiae phospho-Tyr19-Cdc28, total proteins were isolated from growing, unsynchronized cells expressing either an integrated HA-tagged Cdc28 or an integrated Cdc28-HA in which tyrosine 19 was converted to a phenylalanine (Y19F-Cdc28HA). Western blot analyses (A) were carried out as in Fig. 2, first using anti-phospho-Tyr15-Cdc2 antibody (upper panel) and, after washing the filter, using anti-HA antibody 12CA5 (lower panel). To compare the in vivo phosphatase activities of Mih1p in wild-type, cdc55, and mih1 strains (all carrying integrated CDC28:HA genes), cells growing in YPD at 30°C were arrested with HU for 3 h. Cells were then collected by centrifugation, washed, and treated in one of two ways. One-fifth of the cells were resuspended in fresh YPD without HU. At various times thereafter, aliquots of cells were fixed and stained with DAPI. By microscopic examination, the percentages of large-budded cells with two separated nuclei (telophase cells) in each population were determined (B, upper panels). These samples were used to monitor the recovery of cells from S-phase arrest and their subsequent entry into and passage through telophase. The other four-fifths of the cells were resuspended in YPD plus nocodazole so that the cells released from S phase arrest would arrest again at the next metaphase. Aliquots of cells were withdrawn at the time of S-phase release (t = 0) and at various times thereafter (60, 90, 120, 150, and 180 min). At the end of the experiment, cells were stained with DAPI to make certain they were still arrested at metaphase as large-budded cells with a single nucleus. Total cell protein extracts were prepared, and a Western blot analysis was performed using the anti-phospho-Tyr15-Cdc2 antibody (B, middle panels). Subsequently, the filter was washed and reprobed with anti-PSTAIR monoclonal antibody to reveal the amount of Cdc28-HA protein in each lane (B, lower panels). To compare the in vitro Mih1 phosphatase activity of wild-type and cdc55 cells arrested in mitosis, Mih1p was immunoadsorbed to protein A beads from lysates of cells (either cdc55 or CDC55) expressing a MYC-tagged form of Mih1p (see Materials and Methods). As negative controls, similar immunoadsorptions were carried out on CDC55 and cdc55 strains expressing a normal (i.e., nontagged) MIH1 gene. The protein A beads were added to a soluble protein extract prepared from an mih1CDC28:HA strain that had been treated with nocodazole for 3 h (see Materials and Methods for details). The phospho-Y19-Cdc28p in such extracts served as the substrate for the protein A-associated Mih1 phosphatase activity. Following protein A bead addition, the extracts were incubated with agitation at 23°C, and samples were withdrawn at 15-min intervals and, as above, analyzed for phospho-Y19-Cdc28 levels and Cdc28p levels (C). The resulting Western blots were quantitated, and the data for phospho-Y19-Cdc28 were plotted (D). To test the effects of the enzyme assay incubation on the quantity and quality of Mih1p recovered, aliquots of the protein A beads containing Mih1-myc from CDC55 (wt) and cdc55 (c) strains were collected before (one-tenth of the total) and after (one-sixth of the original total) the enzyme assay incubation. The released Mih1p was then subjected to a Western blot analysis (E).

The level of SWE1 kinase is higher in cdc55-null cells. Having determined that decreased levels of Mih1 phosphatase activity are not the likely cause of Y19 hyperphosphorylation in cdc55-null cells, we then measured Swe1p levels in wild-type and cdc55 cells. Swe1p levels normally fluctuate during the cell cycle (31). Therefore, we compared Swe1p levels in wild-type and cdc55-null cells as either asynchronous cultures, HU-arrested (S-phase) cells, or nocodazole-arrested (mitosis) cells (Fig. 4A). In asynchronous cultures, Swe1p levels in cdc55-null cells were slightly higher than that seen in wild-type cells. In HU-arrested cells, both strains had similar amounts of Swe1p. However, in nocodazole-arrested cells, while the wild-type strain had barely detectable amounts of Swe1p, cdc55-null cells had a considerably higher content of Swe1p. The relative levels of Swe1p found in these two strains, whether as asynchronous cultures or as nocodazole-arrested cells, correlated surprisingly well with the relative extent of phosphorylated Cdc28 Y19 found in these two cell types (35) (data not shown), suggesting that it is the increased amount of Swe1p in cdc55-null cells that is the direct cause of Y19 hyperphosphorylation and, consequently, the abnormal budding morphology.

View larger version (35K): [in this window] [in a new window]   FIG. 4.   Metaphase-arrested cdc55-null cells contain more Swe1p than metaphase-arrested wild-type cells. Wild-type and cdc55-null strains expressing an SWE1 gene tagged with 12 copies of the MYC epitope were constructed (see Materials and Methods). Such cells growing in YPD medium at 30°C were collected as unsynchronized cycling cells (lanes 1 and 2), cells arrested in S phase with 0.2 M HU for 3 h (lanes 3 and 4), or cells arrested in mitosis with 20 µg of nocodazole (noc) per ml for 3 h (lanes 5 and 6). Total cell proteins were extracted and analyzed (A) as in Fig. 1, except that we used the monoclonal antibody 9E10 to detect the MYC-tagged protein. The lower half of the gel not used for the protein transfer was stained with Coomassie blue to show the relative protein loading. To see whether the phenomenon of premature sister chromatid separation exhibited by nocodazole-treated cdc55 cells was related to increased Swe1p levels, we determined (B) the relative levels of Swe1p in wild-type (CDC55CDC28), CDC55CDC28VF, cdc55CDC28, and cdc55CDC28VF strains, each expressing a MYC-tagged SWE1 gene (see Materials and Methods for details). Such cells, growing in YPD medium at 30°C, were collected from asynchronous cultures (cycling) or from cultures that had been treated with nocodazole (noc) for either 3 or 6 h; Western analyses were carried out as above. To be able to metaphase arrest wild-type (CDC55) and cdc55-null cells not using drug treatment, we created CDC55 and cdc55 strains expressing SWE1:MYC12 and also carrying the gene cdc23-1. The presence of the latter gene allows inactivation of the APC by raising the temperature of the culture to 35°C. Cultures of the two strains were grown at 25°C in YPD and asynchronous early-log-phase cells were collected (lanes 1 and 2), cultures were treated with HU for 3 h before cell collection (lanes 5 and 6), or cultures were shifted to 35°C for 3 h before cell collection (lanes 3 and 4). Total proteins were collected, and Western blot analyses were carried out to determine Swe1p levels (C). As another way to determine if the excess Swe1p in the metaphase-arrested cdc55-null cells was caused by the lethal effects of the nocodazole or elevated temperature treatments (D), the same wild-type and cdc55-null cells expressing SWE1:MYC12 used in panel A were arrested for 3 h with HU and then washed into fresh YPD medium containing no drug (defined as t = 0). Cells were collected at intervals following the S-phase release for Western blot analysis and for microscopically monitoring cell cycle progression as in Fig. 3. In order to magnify the electrophoretic heterogeneity of the MYC12-tagged Swe1p, SDS-PAGE was carried out using 6% gels rather than the standard 10%. Upper panels, percentages of cells in telophase; middle panels, Swe1p levels; lower panels, loading controls.

cdc55 CDC28

cdc55 CDC28^VF

cdc55 cdc23-1

cdc55 swe1

View larger version (56K): [in this window] [in a new window]   FIG. 5.   SWE1 expression is essential for the morphological defect of cdc55-null cells. cdc55 and swe1 cdc55 strains were grown on YPD plates at 30°C for 1 day and then shifted to 16°C for 2 more days. Cells were harvested, washed, fixed, and then microscopically examined using Nomarski optics. (a) cdc55 cells at 16°C; (b) swe1 cdc55 cells at 16°C.

An increased Swe1p level in mitosis-arrested cdc55-null cells is caused mainly by ectopic protein stabilization. As the level of Swe1p can be regulated during the cell cycle at the transcriptional and posttranscriptional phases (36, 50), as well as the posttranslational level (protein degradation [49]), the increased levels of Swe1p found in cdc55-null cells could be due to alterations in one or more of these processes. If the rate of Swe1 transcription were to be abnormally high and/or the rate of Swe1 mRNA degradation were ectopically decreased at a particular time during the cell cycle, this would have caused an excess of Swe1p to be accumulated. In either case, the manifestation of either of these situations would be an increased steady-state level of Swe1 mRNA at that time when excess Swe1p was present in the cell. To see whether this was the case, we performed a Northern blot analysis on RNA extracted from wild-type and cdc55-null cells that had either been growing asynchronously or been arrested with HU or nocodazole (a portion of samples which were also used for Fig. 4A). These data show (Fig. 6) that as previously demonstrated (50), Swe1 mRNA levels are elevated in S-phase-arrested wild-type cells and considerably depressed in mitosis-arrested cells. They also show that while Swe1 mRNA levels in S-phase-arrested wild-type and cdc55-null cells are indistinguishable, the mRNA level in mitosis-arrested cdc55-null cells is noticeably elevated relative to that in wild-type controls. Assuming no differences in the translation efficiencies of Swe1 mRNA in the two cell types, if the hyperaccumulated Swe1p were only accounted for by the translation of excess mRNA, the relative excesses of mRNA and protein levels in cdc55-null cells versus wild-type cells should be comparable. They are not. In the case of the mRNAs, the measured ratio (i.e., cdc55-null Swe1 mRNA:wild-type Swe1 mRNA) is about 2.5:1; for Swe1p, the measured ratio (from Fig. 4A) is almost 11:1. While elevated Swe1 mRNA can account for some of the excess Swe1p present in cdc55-null cells, it clearly can account for only a small part of it. From these data we cannot tell whether the excess Swe1 mRNA in mitosis-arrested cdc55-null cells is the result of increased Swe1 transcription, a decrease in rate of Swe1 mRNA degradation, or both.

View larger version (75K): [in this window] [in a new window]   FIG. 6.   Swe1 mRNA levels in S- and M-phase-arrested wild-type and cdc55-null cells. Total RNA was extracted from cells that were treated in the same manner as those shown in Fig. 4A. The RNAs were separated on 1% agarose gels and transferred to filters for Northern blot analysis (upper panel). The probe for Swe1 mRNA was generated by random primer labeling a 0.6-kb HindIII/XbaI fragment from the SWE1 ORF. As an indication of relative lane loading, the lower panel shows the ethidium bromide-stained gel prior to transfer. Lanes 1, 3, and 5: wild-type cells as cycling, HU-arrested, and nocodazole-arrested samples, respectively; lanes 2, 4, and 6: cdc55 cells as cycling, HU-arrested, and nocodazole-arrested samples, respectively.

View larger version (30K): [in this window] [in a new window]   FIG. 7.   Normal Swe1p degradation is suppressed in mitosis-arrested cdc55-null cells. To determine the relative rates of degradation of Swe1 mRNA in wild-type and cdc55-null cells, strains expressing chromosomally integrated SWE1-MYC12 genes driven by a GAL1 promoter (see Materials and Methods) were developed. Such cells were grown in YP raffinose to early log phase and then arrested in nocodazole (20 µg/ml) for 3 h. The arrest phenotype was confirmed by microscopic analysis. Galactose was added to each culture to 2% for 30 min to transiently induce SWE1 transcription. Cells were then collected by centrifugation, washed, and resuspended in YPD medium containing nocodazole in order to suppress any further Swe1 mRNA production and to maintain mitotic arrest. Cells were harvested at 0, 25, 50, and 90 min after repression of transcription. Total RNA was extracted, and a Northern analysis (as in Fig. 5) was carried out (a, upper panel). The ethidium bromide-stained agarose gel prior to transfer served as a loading control (a, lower panel). Based on the above results, we measured Swe1p degradation in the following way. Swe1 mRNA was transiently induced as above. Beginning at 50 min after the SWE1 expression had been suppressed by glucose (defined as t = 0), cells were collected at intervals and the levels of Swe1p were determined (b) as in Fig. 3. Western blots were scanned and quantitated, the levels of Swe1p were plotted (c), and the degradation rates of Swe1p in wild-type and cdc55 cells were calculated.

Unregulated PP2A activity is the cause of excess Swe1p accumulation. The absence of Cdc55p, the B-regulatory subunit of the trimeric PP2A, could alter PP2A activity in one of three ways. It could cause the loss of a particular phosphatase activity, it could result in the excessive increase of an already present activity, or it could elicit a novel, i.e., not normally present, phosphatase activity. If the abnormal morphology of cdc55-null cells at low temperature is caused by a loss of a particular PP2A phosphatase activity, one might have expected that the loss of one or both of the PP2A catalytic subunit genes (PPH21 and PPH22) would induce budding defects similar to those exhibited by cdc55-null cells. However, this is not the case (40). On the other hand, if loss of Cdc55p elicits a gain of PP2A function, either a novel one or one already present in the cell, removing one or both of the PP2A catalytic subunit genes in a cdc55-null strain might suppress the morphological defect. Accordingly, we constructed cdc55 pph21, cdc55 pph22, and cdc55 pph21 pph22 strains and observed their growth properties and morphologies at 16°C. While the two double-disruption strains had the same growth properties and morphologies as cdc55-null cells, the triple-knockout strain, while still showing a few cells with abnormal buds at 16°C, was essentially cured of the cdc55-induced morphological phenotype (Fig. 8). These cells did grow more slowly at all temperatures when compared with cdc55-null cells, but no more slowly than pph21 pph22 cells, and their morphologies were also indistinguishable from pph21 pph22 cells (data not shown).

View larger version (67K): [in this window] [in a new window]   FIG. 8.   Disrupting both PPH21 and PPH22 in cdc55-null cells suppresses the morphological defect. Wild-type, cdc55, pph21 cdc55, pph22 cdc55, and pph21 pph22 cdc55 strains were grown on YPD plates at 30°C for 1 day and then were shifted to 16°C for 2 days. Cells were harvested, washed, fixed, and then microscopically examined using Nomarski optics. (a) Wild-type cells; (b) cdc55 cells; (c) pph21 cdc55 cells; (d) pph22 cdc55 cells; (e) pph21 pph22 cdc55 cells.

View larger version (66K): [in this window] [in a new window]   FIG. 9.   Disrupting PPH21 and PPH22 in a cdc55-null strain lowers Swe1p levels in metaphase-arrested cells. Wild-type, cdc55 and pph21 pph22 cdc55 cells (all expressing MYC-tagged SWE1 genes) were grown at 30°C in YPD medium to early log phase. Wild-type and cdc55-null cells were arrested with nocodazole (20 µg/ml) for 3 h. pph21 pph22 cdc55 cells (212255) were treated with nocodazole for 5 h because of the slow growth rate of these cells. The arrest phenotype of all cells was microscopically confirmed prior to their harvest. Western analyses of the proteins isolated from these cells were carried out using anti-MYC antibody to measure Swe1p levels (upper panel) and anti-PSTAIR antibody to detect Cdc28p and Pho85p in order to determine relative protein loading in each lane (lower panel). Lane 1, wild-type cells; lane 2, cdc55 cells; lane 3, pph21 pph22 cdc55 cells.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Given that the budding defect in cdc55-null cells is elicited by the hyperphosphorylation of Cdc28p Y19 and the resulting increased inhibition of this kinase, our results show that this excess phosphorylation is due in large part, if not exclusively, to the ectopic accumulation of Swe1p in these cells. We have shown that elevated Swe1p levels occur because the increased rate of turnover of Swe1p that is normally induced during G₂ in wild-type cells does not occur in cdc55-null cells. These elevated levels of Swe1p in cdc55-null cells apparently maintain continued inhibition of Cdc28 kinase activity at a time when, in wild-type cells, Cdc28 kinase is normally required to regulate the shift from axial to isotropic bud growth (26). Without that switch, abnormal bud development and, hence, the mutant phenotype, can occur.

We believe that while the increased level of Cdc28 Y19 phosphorylation seen in cdc55-null cells is important in inhibiting Cdc28 kinase activity, the increased Swe1p levels themselves can contribute to this inhibition. Swe1p has been shown to be able to inhibit Cdc28p activity through a mechanism independent of Swe1 kinase activity (30). Consistent with this, we found that when Swe1p was overproduced to a level at least 10 times higher than its normal endogenous level, thereby causing a G₂/M arrest, the phosphorylation level on Y19 of Cdc28 in such cells was no higher than that of wild-type controls (data not shown). Furthermore, under these conditions, we found that Cdc28p could be coimmunoprecipitated with Swe1p (data not shown). Thus, Swe1p may bind to Cdc28p and inhibit its activity without increasing the extent of Y19 phosphorylation. In addition, mih1-null cells have a much higher Y19 phosphorylation level than do cdc55-null cells during mitosis (Fig. 2), yet mih1-null cells show only a mildly elongated morphology without a failure of cytokinesis, thus indicating that a high level of Y19 phosphorylation is not sufficient to elicit a cdc55-null-like phenotype. If in cdc55-null cells the abnormal morphology were caused solely by the increased Y19 phosphorylation, then when this phosphorylation level was further increased in a cdc55 mih1 strain, such cells should show an even more abnormal morphology, but that is not what we found (data not shown). The double-null cells appear just like cdc55-null cells, with relatively normal morphology at 30°C and a similar cdc55-null morphology at 16°C (data not shown). We conclude that increasing the Swe1p abundance in cdc55-null cells down-regulates Cdc28 kinase activity, by both Swe1 kinase-dependent and -independent mechanisms.

While we were able to show, by expressing CDC28^VF in cdc55-null cells, that inhibiting premature sister chromatid separation did not in itself prevent Swe1p accumulation in nocodazole-arrested cells (Fig. 4B), we did find the abnormally high levels of Swe1p to be less than that found in cdc55-null cells expressing a normal CDC28 gene. It has been shown that Swe1p positively regulates its own stability (49). A high level of Swe1p inhibits Cdc28 kinase activity, an activity which in itself is required for efficient Swe1p degradation (49). By inhibiting Cdc28 kinase, Swe1p promotes its own accumulation. A CDC28^VF mutant is resistant to Swe1p inhibition, and, consequently, Swe1p is less stable in a strain expressing this gene (49). In cdc55 CDC28^VF cells, this positive feedback loop would be expected to be disrupted, and therefore such cells should have lower levels of Swe1p than are found in cdc55-null cells, which is precisely what we found. However, it is still possible that nocodazole-arrested cdc55-null cells accumulate at a point in mitosis where Swe1p levels normally increase, but no evidence for such an event has been reported (49). Furthermore, if premature sister chromatid separation in cdc55-null cells contributed to Swe1p accumulation significantly, a positive correlation between these two events would be expected. That is not what we observed. It has been shown that sister chromatid separation starts upon entry into mitosis and accumulates during prolonged incubation in nocodazole (35). When the cdc55-null cells were treated with nocodazole for 3 h and 6 h, the percentage of cells with separated sister chromatids should have increased significantly over this period of time (35). However, Swe1p levels did not show a corresponding increase. Instead, they remained the same at these two time points (Fig. 4B). Thus, it seems that the phenomenon of premature sister chromatid separation contributes little to the increased levels of Swe1p found in nocodazole-treated cdc55-null cells.

The step(s) in the regulatory pathway of Swe1p turnover that is adversely affected in cdc55-null cells has not been determined, but our data show that it is the unregulated activity of PP2A phosphatase activity that is responsible, either directly or indirectly, for the alteration in Swe1p degradation rate. If normally there is competition for PP2A regulatory (B or B') subunit binding to AC dimers present in the cell, then one possible source of this unregulated activity in cells missing Cdc55p could be an excess activity of Rts1p-containing PP2A trimers. Consistent with this idea is the fact that overexpression of RTS1 in cdc55-null cells exacerbates the abnormal budding-morphology phenotype (46). Also, rts1 cdc55 strains exhibit no morphological-budding defects at any temperatures (46; H. Yang, unpublished results). While these observations are more consistent with Rts1-directed PP2A activity contributing to the mutant phenotype, other observations support a non-Rts1p-dependent PP2A phosphatase activity being responsible. For example, the strain deleted of the gene encoding Tpd3p, the A (structural) subunit, to which both catalytic and regulatory subunits bind, shows a mutant budding defect not unlike that shown by cdc55-null cells (57). As the coimmunoprecipitation of Rts1p with either of the catalytic subunits, Pph21p or Pph22p, does not occur in the absence of Tpd3p (46; Yang, unpublished results), this suggests that an Rts1p-independent phosphatase activity may elicit the mutant phenotype. Furthermore, as it has been shown that Pph21p and Pph22p can assemble into complexes with proteins other than the classically defined PP2A regulatory and structural subunits (11), it may be the elevated phosphatase activities of such complexes in cdc55-null cells that are responsible for the abnormalities of these cells. Whatever the relative contributions that Rts1-dependent and independent Pph21 and Pph22 phosphatase activities make in eliciting budding defects, what the substrates are that these phosphatases act upon in the cdc55-null strain to produce the mutant phenotype is completely unknown at this time.

Swe1p degradation is regulated at different rates at different times throughout the cell cycle, being highest during G₂ and M (i.e., Swe1p is unstable) and lowest during G₁ and S (48). These changes along with alterations in the Swe1 transcription rate (50) ensure that Swe1p accumulates during G₁, peaking at S/G₂, and then precipitously declines during G₂ and M (31), thereby abolishing any further inhibition of Cdc28 kinase activity. While naturally oscillating, the turnover rate of Swe1p can also be temporarily adjusted in response to cellular checkpoint controls. For example, a temporary disruption of the actin cytoskeleton with latrunculin A during G₁ or S, can bring about a temporary decrease in the Swe1p degradation rate, thereby maintaining inhibition of Cdc28 kinase activity and delaying progression of the cell cycle. The response to actin skeleton disruption does not, however, occur in cells at G₂/M (31). As the abnormal change in Swe1p level in cdc55-null cells is found in mitosis-arrested cells but not S-phase-arrested cells, it would appear that deletion of CDC55 does not simply induce a constitutive actin disruption response.

It has recently been shown that the proteins Hsl1p, a kinase, and Hsl7p, a negative regulator of Swe1p with which it physically interacts, act in concert to target Swe1p for degradation, presumably by phosphorylating it (29, 48). Disruption of either or both of the genes encoding these proteins leads to a stabilization of Swe1p throughout an unperturbed cell cycle (29). Thus, accumulation of Swe1p in cdc55-null cells could conceivably be due to the loss of Hsl1p and/or Hsl7p function. The observation that the electrophoretic mobility shift of Swe1p was not as pronounced in cdc55-null cells upon mitotic entry (Fig. 4D), presumably due to a reduced level of phosphorylation, is reminiscent of what is observed in hsl1 cells (48). This suggests the possibility that Hsl1p kinase activity might be absent or reduced in cdc55 cells. However, we consider this unlikely for the following reasons. Deletion of the gene ELM1, which leads to a loss of Hsl1 kinase function (4, 12), has a synergistic effect with cdc55-null cells. If HSL1 function were already lost in a cdc55-null strain, it is not clear why eliminating ELM1 would necessarily make matters worse. In addition, hsl1 and hsl7 are each synthetically lethal with mih1 (29), while a strain which is cdc55 mih1 is completely viable (H. Yang, unpublished data). While these observations are inconsistent with a total loss of Hsl1 function in cdc55-null cells, further studies on strains mutant for both CDC55 and HSL1 (and possibly HSL7) would be required to establish any causative relationship between Hsl1 kinase function and the Swe1p stabilization seen in cdc55 cells.

Another pathway that could possibly be affected in cdc55-null cells is the one directly responsible for Swe1p degradation, namely Met30-SCF (SKP1-CDC53-F-box protein). Met30-SCF is a protein complex which ubiquitinates and therefore targets Swe1p for proteasome-mediated degradation (20). A genetic interaction between CDC55 and SCF function already exists: a deletion of GRR1, another Met30-like F-box protein known to activate the SCF complex (52), is synthetically lethal with cdc55 (22). It is possibly in this pathway that the uncontrolled PP2A phosphatase activity in cdc55-null cells is having its effect. This certainly is also testable.

We concluded that the contribution of Mih1 phosphatase activity to the higher level of Cdc28 Y19 phosphorylation seen in cdc55-null cells was likely to be minor. The in vivo phosphatase assay showed that, in cdc55-null cells, the MIH1 gene was functioning. Although in mitosis the Mih1p in cdc55 cells is hyperphosphorylated compared to the hypophosphorylated Mih1p in wild-type control cells, their specific phosphatase activities were not much different from each other based on our in vitro measurement. Coupled with the finding that cdc55 cells have amounts of Mih1p similar to those of wild-type cells, Mih1p phosphatase activity in cdc55 cells must be very close to that of wild-type cells.

While we assume that the abnormal budding morphology of cdc55-null cells is due primarily to ectopic Swe1p-induced inhibition of the Cdc28 kinase activity normally required for the apical/isotropic growth switch, is this the only factor? It is likely that elevated Swe1p is necessary, but not sufficient, for the following reasons. First of all, overproducing Swe1p arrests cells at G₂/M with buds emerging from daughter cells (6), a phenotype unlike that of cdc55-null cells. Second, and most important, although Swe1p is essential for the cdc55-null abnormal morphology, Swe1p protein levels don't correlate with the severity of that phenotype (data not shown). The abnormal budding morphology of cdc55-null cells is temperature dependent; at 30°C in glucose medium, the majority of cells look normal, and the abnormal buds that are present are fairly short (46). As the temperature decreases, the frequency of cells with abnormal buds increases, as does the length of the buds. At 16°C essentially all cells have extremely long and abnormally shaped buds. If excess Swe1p were the only factor required for the manifestation of abnormal budding, then one might expect the Swe1p level in those cells at 16°C to be substantially greater than in cells growing at 30°C. This is not the case (data not shown). Thus, it has to be assumed that along with excess Swe1p levels in cdc55-null cells, some other temperature-dependent process must be affected in order to elicit the abnormal budding morphology.

While it is clear that levels of Swe1 mRNA are elevated somewhat in mitosis-arrested cdc55-null cells relative to wild-type controls (Fig. 6), that difference is insufficient to account for the differences seen in Swe1p levels. It is much more likely that an alteration in Swe1p turnover is the major cause of the hyperaccumulation of this protein in cdc55-null cells. As Swe1p has been shown to have, in some unknown way, a positive feedback effect on its own mRNA level (50), the stabilization of Swe1p may well be indirectly the cause of increased Swe1 mRNA levels in mitosis-arrested cdc55-null cells.

While earlier studies, based mainly on the inhibitory effects of okadaic acid, implicated PP2A activity as important in the regulation of mitotic events, we now report that a defined PP2A mutation can alter the turnover properties of a Wee1-like kinase activity during mitosis. This is noteworthy, as PP2A has been implicated as a negative regulator of mitotic entry in fission yeasts and higher eukaryotes and is suspected of doing so through the maintenance of high levels of Cdc2 Y15 phosphorylation. Recently it was reported that in the X. laevis egg, the PP2A-specific inhibitor okadaic acid can induce degradation of Xe-Wee1 at a time when it would normally be stabilized by the DNA replication checkpoint. Furthermore, the ability of okadaic acid to induce mitosis in S phase in the arrested cells was abolished by turning off a Cdc34-dependent ubiquitination function (32). This is certainly consistent with the notion that PP2A activity is required to maintain the SCF ubiquitinating complex activity directed against Xe-Wee1 at a low level until the completion of DNA synthesis. It will be interesting to see whether PP2A activity is also required for the regulation of turnover of Wee1-like kinases in other organisms.