Protein phosphatase 2A regulates MPF activity and sister chromatid cohesion in budding yeast

Background Mitosis is regulated by MPF (maturation promoting factor), the active form of Cdc2/28–cyclin B complexes. Increasing levels of cyclin B abundance and the loss of inhibitory phosphates from Cdc2/28 drives cells into mitosis, whereas cyclin B destruction inactivates MPF and drives cells out of mitosis. Cells with defective spindles are arrested in mitosis by the spindle-assembly checkpoint, which prevents the destruction of mitotic cyclins and the inactivation of MPF. We have investigated the relationship between the spindle-assembly checkpoint, cyclin destruction, inhibitory phosphorylation of Cdc2/28, and exit from mitosis.Results The previously characterized budding yeast mad mutants lack the spindle-assembly checkpoint. Spindle depolymerization does not arrest them in mitosis because they cannot stabilize cyclin B. In contrast, a newly isolated mutant in the budding yeast CDC55 gene, which encodes a protein phosphatase 2A (PP2A) regulatory subunit, shows a different checkpoint defect. In the presence of a defective spindle, these cells separate their sister chromatids and leave mitosis without inducing cyclin B destruction. Despite the persistence of B-type cyclins, cdc55 mutant cells inactivate MPF. Two experiments show that this inactivation is due to inhibitory phosphorylation on Cdc28: phosphotyrosine accumulates on Cdc28 in cdc55Δ cells whose spindles have been depolymerized, and a cdc28 mutant that lacks inhibitory phosphorylation sites on Cdc28 allows spindle defects to arrest cdc55 mutants in mitosis with active MPF and unseparated sister chromatids.Conclusions We conclude that perturbations of protein phosphatase activity allow MPF to be inactivated by inhibitory phosphorylation instead of by cyclin destruction. Under these conditions, sister chromatid separation appears to be regulated by MPF activity rather than by protein degradation. We discuss the role of PP2A and Cdc28 phosphorylation in cell-cycle control, and the possibility that the novel mitotic exit pathway plays a role in adaptation to prolonged activation of the spindle-assembly checkpoint.     

A role for the Pkc1p/Mpk1p kinase cascade in the morphogenesis checkpoint

In many cells the timing of entry into mitosis is controlled by the balance between the activity of inhibitory Wee1-related kinases (Swe1p in budding yeast) and the opposing effect of Cdc25-related phosphatases (Mih1p in budding yeast) that act on the cyclin-dependent kinase Cdc2 (Cdc28p in budding yeast). Wee1 and Cdc25 are key elements in the G2 arrest mediated by diverse checkpoint controls. In budding yeast, a 'morphogenesis checkpoint' that involves Swe1p and Mih1p delays mitotic activation of Cdc28p. Many environmental stresses (such as shifts in temperature or osmolarity) provoke transient depolarization of the actin cytoskeleton, during which bud construction is delayed while cells adapt to environmental conditions. During this delay, the morphogenesis checkpoint halts the cell cycle in G2 phase until actin can repolarize and complete bud construction, thus preventing the generation of binucleate cells. A similar G2 delay can be triggered by mutations or drugs that specifically impair actin organization, indicating that it is probably actin disorganization itself, rather than specific environmental stresses, that triggers the delay. The G2 delay involves stabilization of Swe1p in response to various actin perturbations, although this alone is insufficient to produce a long G2 delay.     

The cdc25 phosphatase is essential for the G2/M phase transition in the basidiomycete yeast Ustilago maydis

Cdc25-related phosphatases reverse the inhibitory phosphorylation of mitotic Cyclin-dependent kinases mediated by Wee1-related kinases, thereby promoting entry into mitosis. In the fission yeast, Schizosaccharomyces pombe, Cdc25 is required for entry into mitosis, while in the budding yeast Saccharomyces cerevisiae, Mih1 (the homologue of Cdc25) is not required for entry into mitosis or for viability. As these differences were linked to the different cell division and growth mechanism of these species, we sought to analyse the roles of Cdc25 in Ustilago maydis, which as S. cerevisiae divides by budding, but relies in a polar growth. This basidiomycete yeast is perfectly suited to analyse the relationships between cell cycle and morphogenesis. We show that U. maydis contains a single Cdc25-related protein, which is essential for growth. Loss of Cdc25 function results in a specific G2 arrest that correlated with high level of Tyr15 phosphorylation of Cdk1. Moreover, we show genetic interactions of cdc25 with wee1 and clb2 that support the notion that in U. maydis Cdc25 counteracts the Wee1-mediated inhibitory phosphorylation of Cdk1-Clb2 complex. Our results supports a model in which inhibitory phosphorylation of Cdk1 is a primary mechanism operating at G2/M transition in this fungus.     

Functional homology among human and fission yeast Cdc14 phosphatases

Budding and fission yeast Cdc14 homologues, a conserved family of serine-threonine phosphatases, play a role in the inactivation of mitotic cyclin-dependent kinases (CDKs) by molecularly distinct mechanisms. Saccharomyces cerevisiae Cdc14 protein phosphatase inactivates CDKs by promoting mitotic cyclin degradation and the accumulation of a CDK inhibitor to allow budding yeast cells to exit from mitosis. Schizosaccharomyces pombe Flp1 phosphatase down-regulates CDK/cyclin activity, controlling the degradation of the Cdc25 tyrosine phosphatase for fission yeast cells to undergo cytokinesis. In the present work, we show that human Cdc14 homologues (hCdc14A and hCdc14B) rescued flp1-deficient fission yeast strains, indicating functional homology. We also show that hCdc14A and B interacted in vivo with S. pombe Cdc25 and that hCdc14A dephosphorylated this mitotic inducer both in vitro and in vivo. Our results support a Cdc14 conserved inhibitory mechanism acting on S. pombe Cdc25 protein and suggest that human cells may regulate Cdc25 in a similar manner to inactivate Cdk1-mitotic cyclin complexes.     

The cytokinin requirement for cell division in cultured Nicotiana plumbaginifolia cells can be satisfied by yeast Cdc25 protein tyrosine phosphatase: implications for mechanisms of cytokinin response and plant development

Cultured cells of Nicotiana plumbaginifolia, when deprived of exogenous cytokinin, arrest in G2 phase prior to mitosis and then contain cyclin-dependent protein kinase (CDK) that is inactive because phosphorylated on tyrosine (Tyr). The action of cytokinin in stimulating the activation of CDK by removal of inhibitory phosphorylation from Tyr is not a secondary downstream consequence of other hormone actions but is the key primary effect of the hormone in its stimulation of cell proliferation, since cytokinin could be replaced by expression of cdc25, which encodes the main Cdc2 (CDK)-Tyr dephosphorylating enzyme of yeast (Saccharomyces cerevisiae). The cdc25 gene, under control of a steroid-inducible promoter, induced a rise in cdc25 mRNA, accumulation of p67(Cdc25) protein, and increase in Cdc25 phosphatase activity that was measured in vitro with Tyr-phosphorylated Cdc2 as substrate. Cdc25 phosphatase activity peaked during mitotic prophase at the time CDK activation was most rapid. Mitosis that was induced by cytokinin also involved increase in endogenous plant CDK Tyr phosphatase activity during prophase, therefore indicating that this is a normal part of plant mitosis. These results suggest a biochemical mechanism for several previously described transgene phenotypes in whole plants and suggest that a primary signal from cytokinin leading to progression through mitosis is the activation of CDK by dephosphorylation of Tyr.     

A cell cycle checkpoint monitors cell morphogenesis in budding yeast

Checkpoint controls are regulatory pathways that inhibit cell cycle progression in cells that have not faithfully completed a prior step in the cell cycle. In the budding yeast Saccharomyces cerevisiae, DNA replication and spindle assembly are monitored by checkpoint controls that prevent nuclear division in cells that have failed to complete these processes. During the normal cell cycle, bud formation is temporally coincident with DNA replication and spindle assembly, and the nucleus divides along the mother-bud axis in mitosis. In this report, we show that inhibition of bud formation also causes a dramatic delay in nuclear division. This allows cells to recover from a transient disruption of cell polarity without becoming binucleate. The delay occurs after DNA replication and spindle assembly, and results from delayed activation of the master cell cycle regulatory kinase, Cdc28. Cdc28 activation is inhibited by phosphorylation of Cdc28 on tyrosine 19, and by delayed accumulation of the B-type cyclins Clb1 and Clb2. These results suggest the existence of a novel checkpoint that monitors cell morphogenesis in budding yeast.     

SPOC alert--when chromosomes get the wrong direction

The asymmetrically dividing budding yeast relies upon the alignment of the mitotic spindle along the mother to daughter cell polarity axis for the fidelity of chromosome segregation during mitosis. In the case of spindle misalignment, a surveillance mechanism named the spindle position checkpoint (SPOC) prevents cells from exiting mitosis through the inhibition of the mitotic exit network (MEN). MEN is a signal transduction pathway that mediates mitotic exit through fully activation of the Cdk-counteracting phosphatase Cdc14. In this mini-review, we briefly describe the mechanisms leading to mitotic exit in budding yeast cells focusing on the control of MEN by the SPOC. In addition, we summarize the recent advances in the molecular understanding of SPOC regulation and discuss whether similar checkpoints may exist in higher eukaryotic cells that undergo asymmetric divisions.     

Stockpiling of Cdc25 during a DNA replication checkpoint arrest in Schizosaccharomyces pombe.

The DNA replication checkpoint couples the onset of mitosis with the completion of S phase. It is clear that in the fission yeast Schizosaccharomyces pombe, operation of this checkpoint requires maintenance of the inhibitory tyrosyl phosphorylation of Cdc2. Cdc25 phosphatase induces mitosis by dephosphorylating tyrosine 15 of Cdc2. In this report, Cdc25 is shown to accumulate to a very high level in cells arrested in S. This shows that mechanisms which modulate the abundance of Cdc25 are unconnected to the DNA replication checkpoint. Using a Cdc2/cyclin B activation assay, we found that Cdc25 activity increased approximately 10-fold during transit through M phase. Cdc25 was activated by phosphorylations that were dependent on Cdc2 activity in vivo. Cdc25 activation was suppressed in cells arrested in G1 and S. However, Cdc25 was more highly modified and appeared to be somewhat more active in S than in G1. This finding might be connected to the fact that progression from G1 to S increases the likelihood that constitutive Cdc25 overproduction will cause inappropriate mitosis.     

Tyrosine Phosphorylation of Cdc2 Is Required for the Replication Checkpoint in Schizosaccharomyces pombe

The DNA replication checkpoint inhibits mitosis in cells that are unable to replicate their DNA, as when nucleotide biosynthesis is inhibited by hydroxyurea. In the fission yeast Schizosaccharomyces pombe, genetic evidence suggests that this checkpoint involves the inhibition of Cdc2 activity through the phosphorylation of tyrosine-15. On the contrary, a recent biochemical study indicated that Cdc2 is in an activated state during a replication checkpoint, suggesting that phosphorylation of Cdc2 on tyrosine-15 is not part of the replication checkpoint mechanism. We have undertaken biochemical and genetic studies to resolve this controversy. We report that the DNA replication checkpoint in S. pombe is abrogated in cells that carry the allele cdc2-Y15F, expressing an unphosphorylatable form of Cdc2. Furthermore, Cdc2 isolated from replication checkpoint-arrested cells can be activated in vitro by Cdc25, the tyrosine phosphatase responsible for dephosphorylating Cdc2 in vivo, to the same extent as Cdc2 isolated from cdc25ts-blocked cells, indicating that hydroxyurea treatment causes Cdc2 activity to be maintained at a low level that is insufficient to induce mitosis. These studies show that inhibitory tyrosine-15 phosphorylation of Cdc2 is essential for the DNA replication checkpoint and suggests that Cdc25, and/or one or both of Wee1 and Mik1, the tyrosine kinases that phosphorylate Cdc2, are regulated by the replication checkpoint.     

Cdk-counteracting phosphatases unlock mitotic exit

Entry into mitosis of the eukaryotic cell cycle is driven by rising cyclin-dependent kinase (Cdk) activity. During exit from mitosis, Cdk activity must again decline. Cdk downregulation by itself, however, is not able to guide mitotic exit, if not a phosphatase reverses mitotic Cdk phosphorylation events. In budding yeast, this role is played by the Cdc14 phosphatase. We are gaining an increasingly detailed picture of its regulation during anaphase, and of the way it orchestrates ordered progression through mitosis. Much less is known about protein dephosphorylation during mitotic exit in organisms other than budding yeast, but evidence is now mounting for crucial contributions of regulated phosphatases also in metazoan cells.     

Phosphorylation of Cdc20/fizzy negatively regulates the mammalian cyclosome/APC in the mitotic checkpoint

The cyclosome/anaphase promoting complex (APC) is a multisubunit ubiquitin ligase that targets mitotic regulators for degradation in exit from mitosis. It is activated at the end of mitosis by phosphorylation and association with the WD-40 protein Cdc20/Fizzy and is then kept active in the G1 phase by association with Cdh1/Hct1. The mitotic checkpoint system that keeps cells with defective spindles from leaving mitosis interacts with Cdc20 and prevents its stimulatory action on the cyclosome. The activity of Cdh1 is negatively regulated by phosphorylation, while the abundance of Cdc20 is cell cycle regulated, with a peak in M-phase. Cdc20 is also phosphorylated in G2/M and in mitotically arrested cells, but the role of phosphorylation remained unknown. Here we show that phosphorylation of Cdc20 by Cdk1/cyclin B abrogates its ability to activate cyclosome/APC from mitotic HeLa cells. A nonphosphorylatable derivative of Cdc20 stimulates cyclin-ubiquitin ligation in extracts from nocodazole-arrested cells to a much greater extent than does wild-type Cdc20. It is suggested that inhibitory phosphorylation of Cdc20/Fizzy may have a role in keeping the cyclosome inactive in early mitosis and under conditions of mitotic checkpoint arrest.     

Many fingers on the mitotic trigger: post-translational regulation of the Cdc25C phosphatase

The mitotic inducer Cdc25 phosphatase controls the activation of Cdc2/cyclin B protein kinase and entry into mitosis in eukaryotic cells. Cdc25C is highly regulated by multiple post-translational modifications within its N-terminal regulatory domain and site-specific protein interactions. Phosphorylation of one inhibitory site targeted by multiple kinases determines the timing of Cdc25C activation and arrests cells in G2 in response to checkpoint, stress, developmental and extracellular signals. In mitosis, phosphorylation of several Ser/Thr residues and Pin1-catalysed peptidyl-proline isomerisation produces activation. Phosphorylation of one activating site is antagonistic to the proximal inhibitory site and maintains Cdc25C activity during mitosis. Phosphorylation and interacting proteins also modulate the nuclear import and export signals on Cdc25C, inducing dramatic changes in its localisation within the cell. Thus, the regulation of Cdc25C activity and localization integrates multiple signals that govern the decision to enter mitosis.     

Many Fingers on the Mitotic Trigger: Post-Translational Regulation of the Cdc25C Phosphatase

AbstractThe mitotic inducer Cdc25 phosphatase controls the activation of Cdc2/cyclin B protein kinase and entry into mitosis in eukaryotic cells. Cdc25C is highly regulated by multiple post-translational modifications within its N-terminal regulatory domain and site-specific protein interactions. Phosphorylation of one inhibitory site targeted by multiple kinases determines the timing of Cdc25C activation and arrests cells in G2 in response to checkpoint, stress, developmental and extracellular signals. In mitosis, phosphorylation of several Ser/Thr residues and Pin1-catalysed peptidyl-proline isomerisation produces activation. Phosphorylation of one activating site is antagonistic to the proximal inhibitory site and maintains Cdc25C activity during mitosis. Phosphorylation and interacting proteins also modulate the nuclear import and export signals on Cdc25C, inducing dramatic changes in its localisation within the cell. Thus, the regulation of Cdc25C activity and localisation integrates multiple signals that govern the decision to enter mitosis.     

Conservation of mechanisms controlling entry into mitosis: budding yeast wee1 delays entry into mitosis and is required for cell size control

BACKGROUND: In fission yeast, the Wee1 kinase delays entry into mitosis until a critical cell size has been reached; however, a similar role for Wee1-related kinases has not been reported in other organisms. SWE1, the budding yeast homolog of wee1, is thought to function in a morphogenesis checkpoint that delays entry into mitosis in response to defects in bud morphogenesis. RESULTS: In contrast to previous studies, we found that budding yeast swe1 Delta cells undergo premature entry into mitosis, leading to birth of abnormally small cells. Additional experiments suggest that conditions that activate the morphogenesis checkpoint may actually be activating a G2/M cell size checkpoint. For example, actin depolymerization is thought to activate the morphogenesis checkpoint by inhibiting bud morphogenesis. However, actin depolymerization also inhibits bud growth, suggesting that it could activate a cell size checkpoint. Consistent with this possibility, we found that actin depolymerization fails to induce a G2/M delay once daughter buds pass a critical size. Other conditions that activate the morphogenesis checkpoint block bud formation, which could also activate a size checkpoint if cell size at G2/M is monitored in the daughter bud. Previous work reported that Swe1 is degraded during G2, which was proposed to account for failure of large-budded cells to arrest in response to actin depolymerization. However, we found that Swe1 is present throughout G2 and undergoes hyperphosphorylation as cells enter mitosis, as found in other organisms. CONCLUSIONS: Our results suggest that the mechanisms known to coordinate entry into mitosis in other organisms have been conserved in budding yeast.     

A functional role for p38 MAPK in modulating mitotic transit in the absence of stress

Although p38 MAPK is known to be activated in response to various environmental stresses and to have inhibitory roles in cell proliferation and tumor progression, its role in cell cycle progression in the absence of stress is unknown in most cell types. In the case of G(2)/M cell cycle control, p38 activation has been shown to trigger a rapid G(2)/M cell cycle checkpoint after DNA damage stress and a spindle checkpoint after microtubule disruption. In the course of our studies, we observed that p38 became actively phosphorylated, and its kinase activity increased transiently during G(2)/M cell cycle transition. Using an immunocytochemistry approach, the active form of p38 was found at the centrosome from late G(2) throughout mitosis, which suggests functional relevance for active p38 protein during mitotic entry. A closer examination reveals that p38 inhibition by pharmacologic inhibitors significantly accelerated the timing of mitotic entry. In addition, long term exposure of the inhibitor enhanced Cdc2 activity. These results indicate that p38 activity during G(2)/M may be involved in a mechanism for fine tuning the initiation of mitosis and perhaps transit of mitosis. Consistent with our previous findings, Cdc25B was phosphorylated on serine 309 at the centrosome during G(2)/M when p38 was active at this site; Cdc25B phosphorylation inhibits Cdc25B activity, and this phosphorylation was found to be p38-dependent. Taken together, our findings suggest that p38 regulates the timing of mitotic entry via modulation of Cdc25B activity under normal nonstress conditions.     

Checkpoint kinase 1 regulates diallyl trisulfide-induced mitotic arrest in human prostate cancer cells

We have shown previously that diallyl trisulfide (DATS), a constituent of processed garlic, inhibits proliferation of PC-3 and DU145 human prostate cancer cells by causing G(2)-M phase cell cycle arrest in association with inhibition of cyclin-dependent kinase 1 activity and hyperphosphorylation of Cdc25C at Ser(216). Here, we report that DATS-treated PC-3 and DU145 cells are also arrested in mitosis as judged by microscopy following staining with anti-alpha-tubulin antibody and 4',6-diamidino-2-phenylindole and flow cytometric analysis of Ser(10) phosphorylation of histone H3. The DATS treatment caused activation of checkpoint kinase 1 and checkpoint kinase 2, which are intermediaries of DNA damage checkpoints and implicated in Ser(216) phosphorylation of Cdc25C. The diallyl trisulfide-induced Ser(216) phosphorylation of Cdc25C as well as mitotic arrest were significantly attenuated by knockdown of check-point kinase 1 protein in both PC-3 and DU145 cells. On the other hand, depletion of checkpoint kinase 2 protein did not have any appreciable effect on G(2) or M phase arrest or Cdc25C phosphorylation caused by diallyl trisulfide. The lack of a role of checkpoint kinase 2 in diallyl trisulfide-induced phosphorylation of Cdc25C or G(2)-M phase cell cycle arrest was confirmed using HCT-15 cells stably transfected with phosphorylation-deficient mutant (T68A mutant) of checkpoint kinase 2. In conclusion, the results of the present study suggest existence of a checkpoint kinase 1-dependent mechanism for diallyl trisulfide-induced mitotic arrest in human prostate cancer cells.     

Cdc14 activates cdc15 to promote mitotic exit in budding yeast

Inactivation of mitotic cyclin-dependent kinases (Cdks) is required for cells to exit mitosis [1] [2]. In the budding yeast Saccharomyces cerevisiae, Cdk inactivation is triggered by the phosphatase Cdc14, which is activated by a complex network of regulatory proteins that includes the protein kinase Cdc15 [3] [4] [5] [6]. Here we show that the ability of Cdc15 to promote mitotic exit is inhibited by phosphorylation. Cdc15 is phosphorylated in vivo at multiple Cdk-consensus sites during most of the cell cycle, but is transiently dephosphorylated in late mitosis. Although phosphorylation appears to have no effect on Cdc15 kinase activity, a non-phosphorylatable mutant of Cdc15 is a more potent stimulator of mitotic exit than wild-type Cdc15, indicating that phosphorylation inhibits Cdc15 function in vivo. Interestingly, inhibitory phosphorylation of Cdc15 is removed by the phosphatase Cdc14 in vitro, and overproduction of Cdc14 leads to Cdc15 dephosphorylation in vivo. Thus, Cdc15 serves both as an activator and substrate of Cdc14. Although this scheme raises the possibility that positive feedback promotes Cdc14 activation, we present evidence that such feedback is not essential for Cdc14 activation in vivo. Instead, Cdc15 dephosphorylation may promote some additional function of Cdc15 that is independent of its effects on Cdc14 activation.     

Regulation of mitotic inhibitor Mik1 helps to enforce the DNA damage checkpoint

The protein kinase Chk1 enforces the DNA damage checkpoint. This checkpoint delays mitosis until damaged DNA is repaired. Chk1 regulates the activity and localization of Cdc25, the tyrosine phosphatase that activates the cdk Cdc2. Here we report that Mik1, a tyrosine kinase that inhibits Cdc2, is positively regulated by the DNA damage checkpoint. Mik1 is required for checkpoint response in strains that lack Cdc25. Long-term DNA damage checkpoint arrest fails in Δmik1 cells. DNA damage increases Mik1 abundance in a Chk1-dependent manner. Ubiquitinated Mik1 accumulates in a proteasome mutant, which indicates that Mik1 normally has a short half-life. Thus, the DNA damage checkpoint might regulate Mik1 degradation. Mik1 protein and mRNA oscillate during the unperturbed cell cycle, with peak amounts detected around S phase. These data indicate that regulation of Mik1 abundance helps to couple mitotic onset to the completion of DNA replication and repair. Coordinated negative regulation of Cdc25 and positive regulation of Mik1 ensure the effective operation of the DNA damage checkpoint.     

Nuclear exclusion of Cdc25 is not required for the DNA damage checkpoint in fission yeast

Maintenance of genome integrity requires a checkpoint that restrains mitosis in response to DNA damage [1]. This checkpoint is enforced by Chk1, a protein kinase that targets Cdc25 [2--7]. Phosphorylated Cdc25 associates with 14-3-3 proteins, which appear to occlude a nuclear localization signal (NLS) and thereby inhibit Cdc25 nuclear import [6, 8--14]. Proficient checkpoint arrest is thought to require Cdc25 nuclear exclusion, although definitive evidence for this model is lacking. We have tested this hypothesis in fission yeast. We show that elimination of an NLS in Cdc25 causes Cdc25 nuclear exclusion and a mitotic delay, as predicted by the model. Attachment of an exogenous NLS forces nuclear inclusion of Cdc25 in damaged cells. However, forced nuclear localization of Cdc25 fails to override the damage checkpoint. Thus, nuclear exclusion of Cdc25 is unnecessary for checkpoint enforcement. We propose that direct inhibition of Cdc25 phosphatase activity by Chk1, as demonstrated in vitro with fission yeast and human Chk1 [15, 16], is sufficient for proficient checkpoint regulation of Cdc25 and may be the primary mechanism of checkpoint enforcement in fission yeast.     

Induction of a mitosis delay and cell lysis by high-level secretion of mouse alpha-amylase from Saccharomyces cerevisiae

Some foreign proteins are produced in yeast in a cell cycle-dependent manner, but the cause of the cell cycle dependency is unknown. In this study, we found that Saccharomyces cerevisiae cells secreting high levels of mouse alpha-amylase have elongated buds and are delayed in cell cycle completion in mitosis. The delayed cell mitosis suggests that critical events during exit from mitosis might be disturbed. We found that the activities of PP2A (protein phosphatase 2A) and MPF (maturation-promoting factor) were reduced in alpha-amylase-oversecreting cells and that these cells showed a reduced level of assembly checkpoint protein Cdc55, compared to the accumulation in wild-type cells. MPF inactivation is due to inhibitory phosphorylation on Cdc28, as a cdc28 mutant which lacks an inhibitory phosphorylation site on Cdc28 prevents MPF inactivation and prevents the defective bud morphology induced by overproduction of alpha-amylase. Our data also suggest that high levels of alpha-amylase may downregulate PPH22, leading to cell lysis. In conclusion, overproduction of heterologous alpha-amylase in S. cerevisiae results in a negative regulation of PP2A, which causes mitotic delay and leads to cell lysis.