The Molecular Function of the Yeast Polo-like Kinase Cdc5 in Cdc14 Release during Early Anaphase

In the budding yeast Saccharomyces cerevisiae, Cdc14 is sequestered within the nucleolus before anaphase entry through its association with Net1/Cfi1, a nucleolar protein. Protein phosphatase PP2A^Cdc55 dephosphorylates Net1 and keeps it as a hypophosphorylated form before anaphase. Activation of the Cdc fourteen early anaphase release (FEAR) pathway after anaphase entry induces a brief Cdc14 release from the nucleolus. Some of the components in the FEAR pathway, including Esp1, Slk19, and Spo12, inactivate PP2A^Cdc55, allowing the phosphorylation of Net1 by mitotic cyclin-dependent kinase (Cdk) (Clb2-Cdk1). However, the function of another FEAR component, the Polo-like kinase Cdc5, remains elusive. Here, we show evidence indicating that Cdc5 promotes Cdc14 release primarily by stimulating the degradation of Swe1, the inhibitory kinase for mitotic Cdk. First, we found that deletion of SWE1 partially suppresses the FEAR defects in cdc5 mutants. In contrast, high levels of Swe1 impair FEAR activation. We also demonstrated that the accumulation of Swe1 in cdc5 mutants is responsible for the decreased Net1 phosphorylation. Therefore, we conclude that the down-regulation of Swe1 protein levels by Cdc5 promotes FEAR activation by relieving the inhibition on Clb2-Cdk1, the kinase for Net1 protein.     

Protein phosphatase 2A (PP2A) promotes anaphase entry after DNA replication stress in budding yeast

DNA replication stress activates the S-phase checkpoint that arrests the cell cycle, but it is poorly understood how cells recover from this arrest. Cyclin-dependent kinase (CDK) and protein phosphatase 2A (PP2A) are key cell cycle regulators, and Cdc55 is a regulatory subunit of PP2A in budding yeast. We found that yeast cells lacking functional PP2A^Cdc55 showed slow growth in the presence of hydroxyurea (HU), a DNA synthesis inhibitor, without obvious viability loss. Moreover, PP2A mutants exhibited delayed anaphase entry and sustained levels of anaphase inhibitor Pds1 after HU treatment. A DNA damage checkpoint Chk1 phosphorylates and stabilizes Pds1. We show that chk1Δ and mutation of the Chk1 phosphorylation sites in Pds1 largely restored efficient anaphase entry in PP2A mutants after HU treatment. In addition, deletion of SWE1, which encodes the inhibitory kinase for CDK or mutation of the Swe1 phosphorylation site in CDK (cdc28F19), also suppressed the anaphase entry delay in PP2A mutants after HU treatment. Our genetic data suggest that Swe1/CDK acts upstream of Pds1. Surprisingly, cdc55Δ showed significant suppression to the viability loss of S-phase checkpoint mutants during DNA synthesis block. Together, our results uncover a PP2A-Swe1-CDK-Chk1-Pds1 axis that promotes recovery from DNA replication stress.     

Budding Yeast Greatwall and Endosulfines Control Activity and Spatial Regulation of PP2ACdc55 for Timely Mitotic Progression

Entry into mitosis is triggered by cyclinB/Cdk1, whose activity is abruptly raised by a positive feedback loop. The Greatwall kinase phosphorylates proteins of the endosulfine family and allows them to bind and inhibit the main Cdk1-counteracting PP2A-B55 phosphatase, thereby promoting mitotic entry. In contrast to most eukaryotic systems, Cdc14 is the main Cdk1-antagonizing phosphatase in budding yeast, while the PP2A^Cdc55 phosphatase promotes, instead of preventing, mitotic entry by participating to the positive feedback loop of Cdk1 activation. Here we show that budding yeast endosulfines (Igo1 and Igo2) bind to PP2A^Cdc55 in a cell cycle-regulated manner upon Greatwall (Rim15)-dependent phosphorylation. Phosphorylated Igo1 inhibits PP2A^Cdc55 activity in vitro and induces mitotic entry in Xenopus egg extracts, indicating that it bears a conserved PP2A-binding and -inhibitory activity. Surprisingly, deletion of IGO1 and IGO2 in yeast cells leads to a decrease in PP2A phosphatase activity, suggesting that endosulfines act also as positive regulators of PP2A in yeast. Consistently, RIM15 and IGO1/2 promote, like PP2A^Cdc55, timely entry into mitosis under temperature-stress, owing to the accumulation of Tyr-phosphorylated Cdk1. In addition, they contribute to the nuclear export of PP2A^Cdc55, which has recently been proposed to promote mitotic entry. Altogether, our data indicate that Igo proteins participate in the positive feedback loop for Cdk1 activation. We conclude that Greatwall, endosulfines, and PP2A are part of a regulatory module that has been conserved during evolution irrespective of PP2A function in the control of mitosis. However, this conserved module is adapted to account for differences in the regulation of mitotic entry in different organisms.     

Dual Regulation of the Mitotic Exit Network (MEN) by PP2A-Cdc55 Phosphatase

Exit from mitosis in budding yeast is triggered by activation of the key mitotic phosphatase Cdc14. At anaphase onset, the protease separase and Zds1 promote the downregulation of PP2A^Cdc55 phosphatase, which facilitates Cdk1-dependent phosphorylation of Net1 and provides the first wave of Cdc14 activity. Once Cdk1 activity starts to decline, the mitotic exit network (MEN) is activated to achieve full Cdc14 activation. Here we describe how the PP2A^Cdc55 phosphatase could act as a functional link between FEAR and MEN due to its action on Bfa1 and Mob1. We demonstrate that PP2A^Cdc55 regulates MEN activation by facilitating Cdc5- and Cdk1-dependent phosphorylation of Bfa1 and Mob1, respectively. Downregulation of PP2A^Cdc55 initiates MEN activity up to Cdc15 by Bfa1 inactivation. Surprisingly, the premature Bfa1 inactivation observed does not entail premature MEN activation, since an additional Cdk1-Clb2 inhibitory signal acting towards Dbf2-Mob1 activity restrains MEN activity until anaphase. In conclusion, we propose a clear picture of how PP2A^Cdc55 functions affect the regulation of various MEN components, contributing to mitotic exit.     

Separase cooperates with Zds1 and Zds2 to activate Cdc14 phosphatase in early anaphase

Completion of mitotic exit and cytokinesis requires the inactivation of mitotic cyclin-dependent kinase (Cdk) activity. A key enzyme that counteracts Cdk during budding yeast mitotic exit is the Cdc14 phosphatase. Cdc14 is inactive for much of the cell cycle, sequestered by its inhibitor Net1 in the nucleolus. At anaphase onset, separase-dependent down-regulation of PP2A^Cdc55 allows phosphorylation of Net1 and consequent Cdc14 release. How separase causes PP2A^Cdc55 down-regulation is not known. Here, we show that two Cdc55-interacting proteins, Zds1 and Zds2, contribute to timely Cdc14 activation during mitotic exit. Zds1 and Zds2 are required downstream of separase to facilitate nucleolar Cdc14 release. Ectopic Zds1 expression in turn is sufficient to down-regulate PP2A^Cdc55 and promote Net1 phosphorylation. These findings identify Zds1 and Zds2 as new components of the mitotic exit machinery, involved in activation of the Cdc14 phosphatase at anaphase onset. Our results suggest that these proteins may act as separase-regulated PP2A^Cdc55 inhibitors.     

Budding Yeast Swe1 Is Involved in the Control of Mitotic Spindle Elongation and Is Regulated by Cdc14 Phosphatase during Mitosis

Cyclin-dependent kinase (Cdk1) activity is required for mitotic entry, and this event is restrained by an inhibitory phosphorylation of the catalytic subunit Cdc28 on a conserved tyrosine (Tyr¹⁹). This modification is brought about by the protein kinase Swe1 that inhibits Cdk1 activation thus blocking mitotic entry. Swe1 levels are regulated during the cell cycle, and they decrease during G₂/M concomitantly to Cdk1 activation, which drives entry into mitosis. However, after mitotic entry, a pool of Swe1 persists, and we collected evidence that it is involved in controlling mitotic spindle elongation. We also describe that the protein phosphatase Cdc14 is implicated in Swe1 regulation; in fact, we observed that Swe1 dephosphorylation in vivo depends on Cdc14 that, in turn, is able to control its subcellular localization. In addition we show that the lack of Swe1 causes premature mitotic spindle elongation and that high levels of Swe1 block mitotic spindle elongation, indicating that Swe1 inhibits this process. Importantly, these effects are not dependent upon the role of in Cdk1 inhibition. These data fit into a model in which Cdc14 binds and inhibits Swe1 to allow timely mitotic spindle elongation.     

Downregulation of PP2A(Cdc55) phosphatase by separase initiates mitotic exit in budding yeast

After anaphase, the high mitotic cyclin-dependent kinase (Cdk) activity is downregulated to promote exit from mitosis. To this end, in the budding yeast S. cerevisiae, the Cdk counteracting phosphatase Cdc14 is activated. In metaphase, Cdc14 is kept inactive in the nucleolus by its inhibitor Net1. During anaphase, Cdk- and Polo-dependent phosphorylation of Net1 is thought to release active Cdc14. How Net1 is phosphorylated specifically in anaphase, when mitotic kinase activity starts to decline, has remained unexplained. Here, we show that PP2A(Cdc55) phosphatase keeps Net1 underphosphorylated in metaphase. The sister chromatid-separating protease separase, activated at anaphase onset, interacts with and downregulates PP2A(Cdc55), thereby facilitating Cdk-dependent Net1 phosphorylation. PP2A(Cdc55) downregulation also promotes phosphorylation of Bfa1, contributing to activation of the "mitotic exit network" that sustains Cdc14 as Cdk activity declines. These findings allow us to present a new quantitative model for mitotic exit in budding yeast.     

DNA replication checkpoint control of Wee1 stability by vertebrate Hsl7

G2/M checkpoints prevent mitotic entry upon DNA damage or replication inhibition by targeting the Cdc2 regulators Cdc25 and Wee1. Although Wee1 protein stability is regulated by DNA-responsive checkpoints, the vertebrate pathways controlling Wee1 degradation have not been elucidated. In budding yeast, stability of the Wee1 homologue, Swe1, is controlled by a regulatory module consisting of the proteins Hsl1 and Hsl7 (histone synthetic lethal 1 and 7), which are targeted by the morphogenesis checkpoint to prevent Swe1 degradation when budding is inhibited. We report here the identification of Xenopus Hsl7 as a positive regulator of mitosis that is controlled, instead, by an entirely distinct checkpoint, the DNA replication checkpoint. Although inhibiting Hsl7 delayed mitosis, Hsl7 overexpression overrode the replication checkpoint, accelerating Wee1 destruction. Replication checkpoint activation disrupted Hsl7-Wee1 interactions, but binding was restored by active polo-like kinase. These data establish Hsl7 as a component of the replication checkpoint and reveal that similar cell cycle control modules can be co-opted for use by distinct checkpoints in different organisms.     

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 Zds proteins control entry into mitosis and target protein phosphatase 2A to the Cdc25 phosphatase

The Wee1 kinase restrains entry into mitosis by phosphorylating and inhibiting cyclin-dependent kinase 1 (Cdk1). The Cdc25 phosphatase promotes entry into mitosis by removing Cdk1 inhibitory phosphorylation. Experiments in diverse systems have established that Wee1 and Cdc25 are regulated by protein phosphatase 2A (PP2A), but a full understanding of the function and regulation of PP2A in entry into mitosis has remained elusive. In budding yeast, entry into mitosis is controlled by a specific form of PP2A that is associated with the Cdc55 regulatory subunit (PP2A(Cdc55)). We show here that related proteins called Zds1 and Zds2 form a tight stoichiometric complex with PP2A(Cdc55) and target its activity to Cdc25 but not to Wee1. Conditional inactivation of the Zds proteins revealed that their function is required primarily at entry into mitosis. In addition, Zds1 undergoes cell cycle-dependent changes in phosphorylation. Together, these observations define a role for the Zds proteins in controlling specific functions of PP2A(Cdc55) and suggest that upstream signals that regulate PP2A(Cdc55) may play an important role in controlling entry into mitosis.     

A phosphatase threshold sets the level of Cdk1 activity in early mitosis in budding yeast

Entry into mitosis is initiated by synthesis of cyclins, which bind and activate cyclin-dependent kinase 1 (Cdk1). Cyclin synthesis is gradual, yet activation of Cdk1 occurs in a stepwise manner: a low level of Cdk1 activity is initially generated that triggers early mitotic events, which is followed by full activation of Cdk1. Little is known about how stepwise activation of Cdk1 is achieved. A key regulator of Cdk1 is the Wee1 kinase, which phosphorylates and inhibits Cdk1. Wee1 and Cdk1 show mutual regulation: Cdk1 phosphorylates Wee1, which activates Wee1 to inhibit Cdk1. Further phosphorylation events inactivate Wee1. We discovered that a specific form of protein phosphatase 2A (PP2A(Cdc55)) opposes the initial phosphorylation of Wee1 by Cdk1. In vivo analysis, in vitro reconstitution, and mathematical modeling suggest that PP2A(Cdc55) sets a threshold that limits activation of Wee1, thereby allowing a low constant level of Cdk1 activity to escape Wee1 inhibition in early mitosis. These results define a new role for PP2A(Cdc55) and reveal a systems-level mechanism by which dynamically opposed kinase and phosphatase activities can modulate signal strength.     

The overlooked greatwall: a new perspective on mitotic control

The role of the dual specificity protein phosphatase, Cdc25, in activating the cyclin-dependent kinase-cyclin B complex (Cdk1-CycB) by overcoming the inhibitory Wee1 kinase is a long-established principle for mitotic entry. Recently, however, evidence has emerged of a regulatory network that facilitates Cdk1-CycB activity by inhibiting the form of protein phosphatase 2A having a B55 regulatory subunit (PP2A-B55). Here, I review the genetic and biochemical evidence for Greatwall kinase and its substrate Endosulphine as the key components of this previously obscure regulatory network. Not only is the inhibition of PP2A-B55 by phospho-endosulphine required to prevent dephosphorylation of Cdk1-CycB substrates until mitotic exit, but it is also required to promote Cdc25 activity and inhibit Wee1 at mitotic entry. I discuss how these alternating states of preferential PP2A-B55 or Cdk1-CycB activity can have an impact upon the regulation of Polo kinase and its ability to bind different partner proteins as mitosis progresses.     

Concerted mechanism of Swe1/Wee1 regulation by multiple kinases in budding yeast

In eukaryotes, entry into mitosis is induced by cyclin B-bound Cdk1, which is held in check by the protein kinase, Wee1. In budding yeast, Swe1 (Wee1 ortholog) is targeted to the bud neck through Hsl1 (Nim1-related kinase) and its adaptor Hsl7, and is hyperphosphorylated prior to ubiquitin-mediated degradation. Here, we show that Hsl1 and Hsl7 are required for proper localization of Cdc5 (Polo-like kinase homolog) to the bud neck and Cdc5-dependent Swe1 phosphorylation. Mitotic cyclin (Clb2)-bound Cdc28 (Cdk1 homolog) directly phosphorylated Swe1 and this modification served as a priming step to promote subsequent Cdc5-dependent Swe1 hyperphosphorylation and degradation. Clb2-Cdc28 also facilitated Cdc5 localization to the bud neck through the enhanced interaction between the Clb2-Cdc28-phosphorylated Swe1 and the polo-box domain of Cdc5. We propose that the concerted action of Cdc28/Cdk1 and Cdc5/Polo on their common substrates is an evolutionarily conserved mechanism that is crucial for effectively triggering mitotic entry and other critical mitotic events.     

A mathematical model of mitotic exit in budding yeast: the role of Polo kinase

Cell cycle progression in eukaryotes is regulated by periodic activation and inactivation of a family of cyclin-dependent kinases (Cdk's). Entry into mitosis requires phosphorylation of many proteins targeted by mitotic Cdk, and exit from mitosis requires proteolysis of mitotic cyclins and dephosphorylation of their targeted proteins. Mitotic exit in budding yeast is known to involve the interplay of mitotic kinases (Cdk and Polo kinases) and phosphatases (Cdc55/PP2A and Cdc14), as well as the action of the anaphase promoting complex (APC) in degrading specific proteins in anaphase and telophase. To understand the intricacies of this mechanism, we propose a mathematical model for the molecular events during mitotic exit in budding yeast. The model captures the dynamics of this network in wild-type yeast cells and 110 mutant strains. The model clarifies the roles of Polo-like kinase (Cdc5) in the Cdc14 early anaphase release pathway and in the G-protein regulated mitotic exit network.     

Adaptation to the spindle checkpoint is regulated by the interplay between Cdc28/Clbs and PP2ACdc55

The spindle checkpoint arrests cells in metaphase until all chromosomes are properly attached to the chromosome segregation machinery. Thereafter, the anaphase promoting complex (APC/C) is activated and chromosome segregation can take place. Cells remain arrested in mitosis for hours in response to checkpoint activation, but not indefinitely. Eventually, they adapt to the checkpoint and proceed along the cell cycle. In yeast, adaptation requires the phosphorylation of APC/C. Here, we show that the protein phosphatase PP2A^Cdc55 dephosphorylates APC/C, thereby counteracting the activity of the mitotic kinase Cdc28. We also observe that the key regulator of Cdc28, the mitotic cyclin Clb2, increases before cells adapt and is then abruptly degraded at adaptation. Adaptation is highly asynchronous and takes place over a range of several hours. Our data suggest the presence of a double negative loop between PP2A^Cdc55 and APC/C^Cdc20 (i.e., a positive feedback loop) that controls APC/C^Cdc20 activity. The circuit could guarantee sustained APC/C^Cdc20 activity after Clb2 starts to be degraded.     

The role of Cdc55 in the spindle checkpoint is through regulation of mitotic exit in Saccharomyces cerevisiae

Cdc55, a B-type regulatory subunit of protein phosphatase 2A, has been implicated in mitotic spindle checkpoint activity and maintenance of sister chromatid cohesion during metaphase. The spindle checkpoint is composed of two independent pathways, one leading to inhibition of the metaphase-to-anaphase transition by checkpoint proteins, including Mad2, and the other to inhibition of mitotic exit by Bub2. We show that Cdc55 is a negative regulator of mitotic exit. A cdc55 mutant, like a bub2 mutant, prematurely releases Cdc14 phosphatase from the nucleolus during spindle checkpoint activation, and premature exit from mitosis indirectly leads to loss of sister chromatid cohesion and inviability in nocodazole. The role of Cdc55 is separable from Bub2 and inhibits release of Cdc14 through a mechanism independent of the known negative regulators of mitotic exit. Epistasis experiments indicate Cdc55 acts either downstream or independent of the mitotic exit network kinase Cdc15. Interestingly, the B-type cyclin Clb2 is partially stable during premature activation of mitotic exit in a cdc55 mutant, indicating mitotic exit is incomplete.     

Cdc55p, the B-type regulatory subunit of protein phosphatase 2A, has multiple functions in mitosis and is required for the kinetochore/spindle checkpoint in Saccharomyces cerevisiae.

Saccharomyces cerevisiae, like most eucaryotic cells, can prevent the onset of anaphase until chromosomes are properly aligned on the mitotic spindle. We determined that Cdc55p (regulatory B subunit of protein phosphatase 2A [PP2A]) is required for the kinetochore/spindle checkpoint regulatory pathway in yeast. ctf13 cdc55 double mutants could not maintain a ctf13-induced mitotic delay, as determined by antitubulin staining and levels of histone H1 kinase activity. In addition, cdc55::LEU2 mutants and tpd3::LEU2 mutants (regulatory A subunit of PP2A) were nocodazole sensitive and exhibited the phenotypes of previously identified kinetochore/spindle checkpoint mutants. Inactivating CDC55 did not simply bypass the arrest that results from inhibiting ubiquitin-dependent proteolysis because cdc16-1 cdc55::LEU2 and cdc23-1 cdc55::LEU2 double mutants arrested normally at elevated temperatures. CDC55 is specific for the kinetochore/spindle checkpoint because cdc55 mutants showed normal sensitivity to gamma radiation and hydroxyurea. The conditional lethality and the abnormal cellular morphogenesis of cdc55::LEU2 were suppressed by cdc28F19, suggesting that the cdc55 phenotypes are dependent on the phosphorylation state of Cdc28p. In contrast, the nocodazole sensitivity of cdc55::LEU2 was not suppressed by cdc28F19. Therefore, the mitotic checkpoint activity of CDC55 (and TPD3) is independent of regulated phosphorylation of Cdc28p. Finally, cdc55::LEU2 suppresses the temperature sensitivity of cdc20-1, suggesting additional roles for CDC55 in mitosis.     

Phosphorylation by Cdc28 activates the Cdc20-dependent activity of the anaphase-promoting complex

Budding yeast initiates anaphase by activating the Cdc20-dependent anaphase-promoting complex (APC). The mitotic activity of Cdc28 (Cdk1) is required to activate this form of the APC, and mutants that are impaired in mitotic Cdc28 function have difficulty leaving mitosis. This defect can be explained by a defect in APC phosphorylation, which depends on mitotic Cdc28 activity in vivo and can be catalyzed by purified Cdc28 in vitro. Mutating putative Cdc28 phosphorylation sites in three components of the APC, Cdc16, Cdc23, and Cdc27, makes the APC resistant to phosphorylation both in vivo and in vitro. The nonphosphorylatable APC has normal activity in G1, but its mitotic, Cdc20-dependent activity is compromised. These results show that Cdc28 activates the APC in budding yeast to trigger anaphase. Previous reports have shown that the budding yeast Cdc5 homologue, Plk, can also phosphorylate and activate the APC in vitro. We show that, like cdc28 mutants, cdc5 mutants affect APC phosphorylation in vivo. However, although Cdc5 can phosphorylate Cdc16 and Cdc27 in vitro, this in vitro phosphorylation does not occur on in vivo sites of phosphorylation.     

Switches and latches: a biochemical tug-of-war between the kinases and phosphatases that control mitosis

Activation of the cyclin-dependent kinase (Cdk1) cyclin B (CycB) complex (Cdk1:CycB) in mitosis brings about a remarkable extent of protein phosphorylation. Cdk1:CycB activation is switch-like, controlled by two auto-amplification loops--Cdk1:CycB activates its activating phosphatase, Cdc25, and inhibits its inhibiting kinase, Wee1. Recent experimental evidence suggests that parallel to Cdk1:CycB activation during mitosis, there is inhibition of its counteracting phosphatase activity. We argue that the downregulation of the phosphatase is not just a simple latch that suppresses futile cycles of phosphorylation/dephosphorylation during mitosis. Instead, we propose that phosphatase regulation creates coherent feed-forward loops and adds extra amplification loops to the Cdk1:CycB regulatory network, thus forming an integral part of the mitotic switch. These network motifs further strengthen the bistable characteristic of the mitotic switch, which is based on the antagonistic interaction of two groups of proteins: M-phase promoting factors (Cdk1:CycB, Cdc25, Greatwall and Endosulfine/Arpp19) and interphase promoting factors (Wee1, PP2A-B55 and a Greatwall counteracting phosphatase, probably PP1). The bistable character of the switch implies the existence of a CycB threshold for entry into mitosis. The end of G2 phase is determined by the point where CycB level crosses the CycB threshold for Cdk1 activation.     

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.