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.     

The Morphogenesis Checkpoint in Saccharomyces cerevisiae: Cell Cycle Control of Swe1p Degradation by Hsl1p and Hsl7p

In Saccharomyces cerevisiae, the Wee1 family kinase Swe1p is normally stable during G(1) and S phases but is unstable during G(2) and M phases due to ubiquitination and subsequent degradation. However, perturbations of the actin cytoskeleton lead to a stabilization and accumulation of Swe1p. This response constitutes part of a morphogenesis checkpoint that couples cell cycle progression to proper bud formation, but the basis for the regulation of Swe1p degradation by the morphogenesis checkpoint remains unknown. Previous studies have identified a protein kinase, Hsl1p, and a phylogenetically conserved protein of unknown function, Hsl7p, as putative negative regulators of Swe1p. We report here that Hsl1p and Hsl7p act in concert to target Swe1p for degradation. Both proteins are required for Swe1p degradation during the unperturbed cell cycle, and excess Hsl1p accelerates Swe1p degradation in the G(2)-M phase. Hsl1p accumulates periodically during the cell cycle and promotes the periodic phosphorylation of Hsl7p. Hsl7p can be detected in a complex with Swe1p in cell lysates, and the overexpression of Hsl7p or Hsl1p produces an effective override of the G(2) arrest imposed by the morphogenesis checkpoint. These findings suggest that Hsl1p and Hsl7p interact directly with Swe1p to promote its recognition by the ubiquitination complex, leading ultimately to its destruction.     

Determinants of Swe1p degradation in Saccharomyces cerevisiae

Swe1p, the sole Wee1-family kinase in Saccharomyces cerevisiae, is synthesized during late G1 and is then degraded as cells proceed through the cell cycle. However, Swe1p degradation is halted by the morphogenesis checkpoint, which responds to insults that perturb bud formation. The Swe1p stabilization promotes cell cycle arrest through Swe1p-mediated inhibitory phosphorylation of Cdc28p until the cells can recover from the perturbation and resume bud formation. Swe1p degradation involves the relocalization of Swe1p from the nucleus to the mother-bud neck, and neck targeting requires the Swe1p-interacting protein Hsl7p. In addition, Swe1p degradation is stimulated by its substrate, cyclin/Cdc28p, and Swe1p is thought to be a target of the ubiquitin ligase SCF(Met30) acting with the ubiquitin-conjugating enzyme Cdc34p. The basis for regulation of Swe1p degradation by the morphogenesis checkpoint remains unclear, and in order to elucidate that regulation we have dissected the Swe1p degradation pathway in more detail, yielding several novel findings. First, we show here that Met30p (and by implication SCF(Met30)) is not, in fact, required for Swe1p degradation. Second, cyclin/Cdc28p does not influence Swe1p neck targeting, but can directly phosphorylate Swe1p, suggesting that it acts downstream of neck targeting in the Swe1p degradation pathway. Third, a screen for functional but nondegradable mutants of SWE1 identified two small regions of Swe1p that are key to its degradation. One of these regions mediates interaction of Swe1p with Hsl7p, showing that the Swe1p-Hsl7p interaction is critical for Swe1p neck targeting and degradation. The other region did not appear to affect interactions with known Swe1p regulators, suggesting that other as-yet-unknown regulators exist.     

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.     

Mathematical model of the morphogenesis checkpoint in budding yeast

The morphogenesis checkpoint in budding yeast delays progression through the cell cycle in response to stimuli that prevent bud formation. Central to the checkpoint mechanism is Swe1 kinase: normally inactive, its activation halts cell cycle progression in G2. We propose a molecular network for Swe1 control, based on published observations of budding yeast and analogous control signals in fission yeast. The proposed Swe1 network is merged with a model of cyclin-dependent kinase regulation, converted into a set of differential equations and studied by numerical simulation. The simulations accurately reproduce the phenotypes of a dozen checkpoint mutants. Among other predictions, the model attributes a new role to Hsl1, a kinase known to play a role in Swe1 degradation: Hsl1 must also be indirectly responsible for potent inhibition of Swe1 activity. The model supports the idea that the morphogenesis checkpoint, like other checkpoints, raises the cell size threshold for progression from one phase of the cell cycle to the next.     

Clb2 and the APC/CCdh1 regulate Swe1 stability

Swe1/Wee1 regulates mitotic entry by inhibiting Clb2-Cdk1 and its accumulation is involved in stress induced G2 arrest. The APC/C^Cdh1 substrates Cdc5, Clb2 and Hsl1 regulate Swe1 degradation. We observed that clb2Dcdh1D double mutant S. cerevisiae does not express any detectable levels of Swe1, presumably due to its constitutive degradation. This effect of Cdh1 inactivation is due to stabilization of Cdc5 and Hsl1, as expression of the non-degradable Cdc5^T29A in clb2D cells prevented Swe1 accumulation. Strikingly, expression of non-degradable Hsl1^mdb/mkb prevented Swe1 accumulation even in wild type Clb2 cells. Interestingly Swe1 accumulation could be reconstituted in all these mutants by eliciting a replication fork stress with hydroxyurea. Cells expressing the Clb2^ME mutant, that cannot bind Swe1, behaved like clb2D cells, and failed to accumulate Swe1 in the absence of Cdh1 or the presence of Cdc5^T29A. This suggests that for Swe1 to accumulate it must interact with Clb2. We further show that in the absence of Clb2, Hsl1 is no longer essential for Swe1 degradation. We hypothesize that Clb2-Cdk1 protects Swe1 from premature degradation until its Hsl1 mediated de-protection, which enables its Cdc5 mediated degradation. Swe1 levels are thus regulated by monitoring the levels of three major mitotic regulators.     

Hsl7 Localizes to a Septin Ring and Serves as an Adapter in a Regulatory Pathway That Relieves Tyrosine Phosphorylation of Cdc28 Protein Kinase in Saccharomyces cerevisiae

Successful mitosis requires faithful DNA replication, spindle assembly, chromosome segregation, and cell division. In the budding yeast Saccharomyces cerevisiae, the G(2)-to-M transition requires activation of Clb-bound forms of the protein kinase, Cdc28. These complexes are held in an inactive state via phosphorylation of Tyr19 in the ATP-binding loop of Cdc28 by the Swe1 protein kinase. The HSL1 and HSL7 gene products act as negative regulators of Swe1. Hsl1 is a large (1,518-residue) protein kinase with an N-terminal catalytic domain and a very long C-terminal extension. Hsl1 localizes to the incipient site of cytokinesis in the bud neck in a septin-dependent manner; however, the function of Hsl7 was not previously known. Using both indirect immunofluorescence with anti-Hsl7 antibodies and a fusion of Hsl7 to green fluorescent protein, we found that Hsl7 also localizes to the bud neck, congruent with the septin ring that faces the daughter cell. Both Swe1 and a segment of the C terminus of Hsl1 (which has no sequence counterpart in two Hsl1-related protein kinases, Gin4 and Kcc4) were identified as gene products that interact with Hsl7 in a two-hybrid screen of a random S. cerevisiae cDNA library. Hsl7 plus Swe1 and Hsl7 plus Hsl1 can be coimmunoprecipitated from extracts of cells overexpressing these proteins, confirming that Hsl7 physically associates with both partners. Also consistent with the two-hybrid results, Hsl7 coimmunoprecipitates with full-length Hsl1 less efficiently than with a C-terminal fragment of Hsl1. Moreover, Hsl7 does not localize to the bud neck in an hsl1Delta mutant, whereas Hsl1 is localized normally in an hsl7Delta mutant. Phosphorylation and ubiquitinylation of Swe1, preludes to its destruction, are severely reduced in cells lacking either Hsl1 or Hsl7 (or both), as judged by an electrophoretic mobility shift assay. Collectively, these data suggest that formation of the septin rings provides sites for docking Hsl1, exposing its C terminus and thereby permitting recruitment of Hsl7. Hsl7, in turn, presents its cargo of bound Swe1, allowing phosphorylation by Hsl1. Thus, Hsl1 and Hsl7 promote proper timing of cell cycle progression by coupling septin ring assembly to alleviation of Swe1-dependent inhibition of Cdc28. Furthermore, like septins and Hsl1, homologs of Hsl7 are found in fission yeast, flies, worms, and humans, suggesting that its function in this control mechanism may be conserved in all eukaryotes.     

Dynamic Localization of the Swe1 Regulator Hsl7 During the Saccharomyces cerevisiae Cell Cycle

In Saccharomyces cerevisiae, entry into mitosis requires activation of the cyclin-dependent kinase Cdc28 in its cyclin B (Clb)-associated form. Clb-bound Cdc28 is susceptible to inhibitory tyrosine phosphorylation by Swe1 protein kinase. Swe1 is itself negatively regulated by Hsl1, a Nim1-related protein kinase, and by Hsl7, a presumptive protein-arginine methyltransferase. In vivo all three proteins localize to the bud neck in a septin-dependent manner, consistent with our previous proposal that formation of Hsl1-Hsl7-Swe1 complexes constitutes a checkpoint that monitors septin assembly. We show here that Hsl7 is phosphorylated by Hsl1 in immune-complex kinase assays and can physically associate in vitro with either Hsl1 or Swe1 in the absence of any other yeast proteins. With the use of both the two-hybrid method and in vitro binding assays, we found that Hsl7 contains distinct binding sites for Hsl1 and Swe1. A differential interaction trap approach was used to isolate four single-site substitution mutations in Hsl7, which cluster within a discrete region of its N-terminal domain, that are specifically defective in binding Hsl1. When expressed in hsl7Delta cells, each of these Hsl7 point mutants is unable to localize at the bud neck and cannot mediate down-regulation of Swe1, but retains other functions of Hsl7, including oligomerization and association with Swe1. GFP-fusions of these Hsl1-binding defective Hsl7 proteins localize as a bright perinuclear dot, but never localize to the bud neck; likewise, in hsl1Delta cells, a GFP-fusion to wild-type Hsl7 or native Hsl7 localizes to this dot. Cell synchronization studies showed that, normally, Hsl7 localizes to the dot, but only in cells in the G1 phase of the cell cycle. Immunofluorescence analysis and immunoelectron microscopy established that the dot corresponds to the outer plaque of the spindle pole body (SPB). These data demonstrate that association between Hsl1 and Hsl7 at the bud neck is required to alleviate Swe1-imposed G2-M delay. Hsl7 localization at the SPB during G1 may play some additional role in fine-tuning the coordination between nuclear and cortical events before mitosis.     

Roles of the mitotic inhibitors Wee1 and Mik1 in the G(2) DNA damage and replication checkpoints

The G(2) DNA damage and DNA replication checkpoints in many organisms act through the inhibitory phosphorylation of Cdc2 on tyrosine-15. This phosphorylation is catalyzed by the Wee1/Mik1 family of kinases. However, the in vivo role of these kinases in checkpoint regulation has been unclear. We show that, in the fission yeast Schizosaccharomyces pombe, Mik1 is a target of both checkpoints and that the regulation of Mik1 is, on its own, sufficient to delay mitosis in response to the checkpoints. Mik1 appears to have two roles in the DNA damage checkpoint; one in the establishment of the checkpoint and another in its maintenance. In contrast, Wee1 does not appear to be involved in the establishment of either checkpoint.     

Molecular Dissection of the Checkpoint Kinase Hsl1p

Cell shape can influence cell behavior. In Saccharomyces cerevisiae, bud emergence can influence cell cycle progression via the morphogenesis checkpoint. This surveillance pathway ensures that mitosis always follows bud formation by linking degradation of the mitosis-inhibitory kinase Swe1p (Wee1) to successful bud emergence. A crucial component of this pathway is the checkpoint kinase Hsl1p, which is activated upon bud emergence and promotes Swe1p degradation. We have dissected the large nonkinase domain of Hsl1p by using evolutionary conservation as a guide, identifying regions important for Hsl1p localization, function, and regulation. An autoinhibitory motif restrains Hsl1p activity when it is not properly localized to the mother-bud neck. Hsl1p lacking this motif is active as a kinase regardless of the assembly state of cytoskeletal septin filaments. However, the active but delocalized Hsl1p cannot promote Swe1p down-regulation, indicating that localization is required for Hsl1p function as well as Hsl1p activation. We also show that the septin-mediated Hsl1p regulation via the novel motif operates in parallel to a previously identified Hsl1p activation pathway involving phosphorylation of the Hsl1p kinase domain. We suggest that Hsl1p responds to alterations in septin organization, which themselves occur in response to the local geometry of the cell cortex.     

Three Different Pathways Prevent Chromosome Segregation in the Presence of DNA Damage or Replication Stress in Budding Yeast

A surveillance mechanism, the S phase checkpoint, blocks progression into mitosis in response to DNA damage and replication stress. Segregation of damaged or incompletely replicated chromosomes results in genomic instability. In humans, the S phase checkpoint has been shown to constitute an anti-cancer barrier. Inhibition of mitotic cyclin dependent kinase (M-CDK) activity by Wee1 kinases is critical to block mitosis in some organisms. However, such mechanism is dispensable in the response to genotoxic stress in the model eukaryotic organism Saccharomyces cerevisiae. We show here that the Wee1 ortholog Swe1 does indeed inhibit M-CDK activity and chromosome segregation in response to genotoxic insults. Swe1 dispensability in budding yeast is the result of a redundant control of M-CDK activity by the checkpoint kinase Rad53. In addition, our results indicate that Swe1 is an effector of the checkpoint central kinase Mec1. When checkpoint control on M-CDK and on Pds1/securin stabilization are abrogated, cells undergo aberrant chromosome segregation.     

The checkpoint kinase Hsl1p is activated by Elm1p-dependent phosphorylation

Saccharomyces cerevisiae cells growing in the outdoor environment must adapt to sudden changes in temperature and other variables. Many such changes trigger stress responses that delay bud emergence until the cells can adapt. In such circumstances, the morphogenesis checkpoint delays mitosis until a bud has been formed. Mitotic delay is due to the Wee1 family mitotic inhibitor Swe1p, whose degradation is linked to bud emergence by the checkpoint kinase Hsl1p. Hsl1p is concentrated at the mother-bud neck through association with septin filaments, and it was reported that Hsl1p activation involved relief of autoinhibition in response to septin interaction. Here we challenge the previous identification of an autoinhibitory domain and show instead that Hsl1p activation involves the phosphorylation of threonine 273, promoted by the septin-associated kinase Elm1p. We identified elm1 mutants in a screen for defects in Swe1p degradation and show that a phosphomimic T273E mutation in HSL1 bypasses the need for Elm1p in this pathway.     

Eavesdropping on the cytoskeleton: progress and controversy in the yeast morphogenesis checkpoint

The morphogenesis checkpoint provides a link between bud formation and mitosis in yeast. In this pathway, insults affecting the actin or septin cytoskeleton trigger a cell cycle arrest, mediated by the Wee1 homolog Swe1p, which catalyzes the inhibitory phosphorylation of the mitosis-promoting cyclin-dependent kinase (CDK) on a conserved tyrosine residue. Analyses of Swe1p phosphorylation have mapped 61 sites targeted by CDKs and Polo-related kinases, which control both Swe1p activity and Swe1p degradation. Although the sites themselves are not evolutionarily conserved, the control of Swe1p degradation exhibits many conserved features, and is linked to DNA-responsive checkpoints in vertebrate cells. At the 'sensing' end of the checkpoint, recent work has begun to shed light on how septins are organized and how they impact Swe1p regulators. However, the means by which Swe1p responds to actin perturbations once a bud has formed remains controversial.     

Cdc25 Inhibited In Vivo and In Vitro by Checkpoint Kinases Cds1 and Chk1

In the fission yeast Schizosaccharomyces pombe, the protein kinase Cds1 is activated by the S-M replication checkpoint that prevents mitosis when DNA is incompletely replicated. Cds1 is proposed to regulate Wee1 and Mik1, two tyrosine kinases that inhibit the mitotic kinase Cdc2. Here, we present evidence from in vivo and in vitro studies, which indicates that Cds1 also inhibits Cdc25, the phosphatase that activates Cdc2. In an in vivo assay that measures the rate at which Cdc25 catalyzes mitosis, Cds1 contributed to a mitotic delay imposed by the S-M replication checkpoint. Cds1 also inhibited Cdc25-dependent activation of Cdc2 in vitro. Chk1, a protein kinase that is required for the G2-M damage checkpoint that prevents mitosis while DNA is being repaired, also inhibited Cdc25 in the in vitro assay. In vitro, Cds1 and Chk1 phosphorylated Cdc25 predominantly on serine-99. The Cdc25 alanine-99 mutation partially impaired the S-M replication and G2-M damage checkpoints in vivo. Thus, Cds1 and Chk1 seem to act in different checkpoint responses to regulate Cdc25 by similar mechanisms.     

Feedback control of Swe1p degradation in the yeast morphogenesis checkpoint

Saccharomyces cerevisiae cells exposed to a variety of physiological stresses transiently delay bud emergence or bud growth. To maintain coordination between bud formation and the cell cycle in such circumstances, the morphogenesis checkpoint delays nuclear division via the mitosis-inhibitory Wee1-family kinase, Swe1p. Swe1p is degraded during G2 in unstressed cells but is stabilized and accumulates following stress. Degradation of Swe1p is preceded by its recruitment to the septin scaffold at the mother-bud neck, mediated by the Swe1p-binding protein Hsl7p. Following osmotic shock or actin depolymerization, Swe1p is stabilized, and previous studies suggested that this was because Hsl7p was no longer recruited to the septin scaffold following stress. However, we now show that Hsl7p is in fact recruited to the septin scaffold in stressed cells. Using a cyclin-dependent kinase (CDK) mutant that is immune to checkpoint-mediated inhibition, we show that Swe1p stabilization following stress is an indirect effect of CDK inhibition. These findings demonstrate the physiological importance of a positive-feedback loop in which Swe1p activity inhibits the CDK, which then ceases to target Swe1p for degradation. They also highlight the difficulty in disentangling direct checkpoint pathways from the effects of positive-feedback loops active at the G2/M transition.     

Hsl1p, a Swe1p inhibitor, is degraded via the anaphase-promoting complex

Ubiquitination and subsequent degradation of critical cell cycle regulators is a key mechanism exploited by the cell to ensure an irreversible progression of cell cycle events. The anaphase-promoting complex (APC) is a ubiquitin ligase that targets proteins for degradation by the 26S proteasome. Here we identify the Hsl1p protein kinase as an APC substrate that interacts with Cdc20p and Cdh1p, proteins that mediate APC ubiquitination of protein substrates. Hsl1p is absent in G(1), accumulates as cells begin to bud, and disappears in late mitosis. Hsl1p is stabilized by mutations in CDH1 and CDC23, both of which result in compromised APC activity. Unlike Hsl1p, Gin4p and Kcc4p, protein kinases that have sequence homology to Hsl1p, were stable in G(1)-arrested cells containing active APC. Mutation of a destruction box motif within Hsl1p (Hsl1p(db-mut)) stabilized Hsl1p. Interestingly, this mutation also disrupted the Hsl1p-Cdc20p interaction and reduced the association between Hsl1p and Cdh1p in coimmunoprecipitation studies. These findings suggest that the destruction box motif is required for Cdc20p and, to a lesser extent, for Cdh1p to target Hsl1p to the APC for ubiquitination. Hsl1p has been previously shown to inhibit Swe1p, a protein kinase that negatively regulates the cyclin-dependent kinase Cdc28p, by promoting Swe1p degradation via SCF(Met30) in a bud morphogenesis checkpoint. Results of the present work indicate that Hsl1p is degraded in an APC-dependent manner and suggest a link between the SCF (Skp1-cullin-F box) and APC-proteolytic systems that may help to coordinate the proper progression of cell cycle events.     

Phosphorylation-Independent Inhibition of Cdc28p by the Tyrosine Kinase Swe1p in the Morphogenesis Checkpoint

The morphogenesis checkpoint in budding yeast delays cell cycle progression in G(2) when the actin cytoskeleton is perturbed, providing time for cells to complete bud formation prior to mitosis. Checkpoint-induced G(2) arrest involves the inhibition of the master cell cycle regulatory cyclin-dependent kinase, Cdc28p, by the Wee1 family kinase Swe1p. Results of experiments using a nonphosphorylatable CDC28(Y19F) allele suggested that the checkpoint stimulated two inhibitory pathways, one that promoted phosphorylation at tyrosine 19 (Y19) and a poorly characterized second pathway that did not require Cdc28p Y19 phosphorylation. We present the results from a genetic screen for checkpoint-defective mutants that led to the repeated isolation of the dominant CDC28(E12K) allele that is resistant to Swe1p-mediated inhibition. Comparison of this allele with the nonphosphorylatable CDC28(Y19F) allele suggested that Swe1p is still able to inhibit CDC28(Y19F) in a phosphorylation-independent manner and that both the Y19 phosphorylation-dependent and -independent checkpoint pathways in fact reflect Swe1p inhibition of Cdc28p. Remarkably, we found that a Swe1p mutant lacking catalytic activity could significantly delay the cell cycle in vivo during a physiological checkpoint response, even when expressed at single copy. The finding that a Wee1 family kinase expressed at physiological levels can inhibit a nonphosphorylatable cyclin-dependent kinase has broad implications for many checkpoint studies using such mutants in other organisms.     

Zds2p regulates Swe1p-dependent polarized cell growth in Saccharomyces cerevisiae via a novel Cdc55p interaction domain

Deletion of the paralogs ZDS1 and ZDS2 in the budding yeast Saccharomyces cerevisiae causes a mis-regulation of polarized cell growth. Here we show a function for these genes as regulators of the Swe1p (Wee1p) kinase-dependent G2/M checkpoint. We identified a conserved domain in the C-terminus of Zds2p consisting of amino acids 813-912 (hereafter referred to as ZH4 for Zds homology 4) that is required for regulation of Swe1p-dependent polarized bud growth. ZH4 is shown by protein affinity assays to be necessary and sufficient for interaction with Cdc55p, a regulatory subunit of protein phosphatase 2A (PP2A). We hypothesized that the Zds proteins are in a pathway that negatively regulates the Swe1p-dependent G2/M checkpoint via Cdc55p. Supporting this model, deletion of CDC55 rescues the aberrant bud morphology of a zds1Δzds2Δ strain. We also show that expression of ZDS1 or ZDS2 from a strong galactose-inducible promoter can induce mitosis even when the Swe1p-dependent G2/M checkpoint is activated by mis-organization of the actin cytoskeleton. This negative regulation requires the CDC55 gene. Together these data indicate that the Cdc55p/Zds2p module has a function in the regulation of the Swe1p-dependent G2/M checkpoint.     

Monitoring the Cell Cycle by Multi-Kinase-Dependent Regulation of Swe1/Wee1 in Budding Yeast

In eukaryotes, G2/M transition is induced by the activation of cyclin B-bound Cdk1, which is held in check by the protein kinase, Wee1. Recent advances in our understanding of mitotic entry in budding yeast has revealed that these cells utilize the level of Swe1 (Wee1 ortholog) phosphorylation as a means of monitoring cell cycle progression and of coordinating morphogenetic events with mitotic entry. Swe1 is phosphorylated by at least three distinct kinases at different stages of the cell cycle. This cumulative phosphorylation leads to the hyperphosphorylation and degradation of Swe1 through ubiquitin-mediated proteolysis. Thus, Swe1 functions as an important cell cycle modulator that integrates multiple upstream signals from prior cell cycle events before its ultimate degradation permits passage into mitosis.     

Control of Swe1p degradation by the morphogenesis checkpoint.

In the budding yeast Saccharomyces cerevisiae, a cell cycle checkpoint coordinates mitosis with bud formation. Perturbations that transiently depolarize the actin cytoskeleton cause delays in bud formation, and a 'morphogenesis checkpoint' detects the actin perturbation and imposes a G2 delay through inhibition of the cyclin-dependent kinase, Cdc28p. The tyrosine kinase Swe1p, homologous to wee1 in fission yeast, is required for the checkpoint-mediated G2 delay. In this report, we show that Swe1p stability is regulated both during the normal cell cycle and in response to the checkpoint. Swe1p is stable during G1 and accumulates to a peak at the end of S phase or in early G2, when it becomes unstable and is degraded rapidly. Destabilization of Swe1p in G2 and M phase depends on the activity of Cdc28p in complexes with B-type cyclins. Several different perturbations of actin organization all prevent Swe1p degradation, leading to the persistence or further accumulation of Swe1p, and cell cycle delay in G2.