Cyclin-specific START events and the G1-phase specificity of arrest by mating factor in budding yeast

The START cell cycle transition in the budding yeast Saccharomyces cerevisiae is catalyzed by the Cdc28 cyclin-dependent kinase associated with Cln-type cyclins. Since ectopic expression of the B-type cyclin CLB5 can efficiently rescue the inviability that results from CLN depletion, we tested the specificity of the CLN and CLB classes of cyclins for promoting START-associated events. Several aspects of the regulation of the mating factor response were compared for cells in which START activity was provided by either Cln-cyclins or Clb5. Unlike Cln1 and Cln2, high level expression of Clb5 was unable to repress the activity of the mating factor response pathway at START. Downregulation of Far1 protein at START is normal in cln−GAL1::CLB5 cells. Even though the Clb5-Cdc28 kinase activity in cln−GAL1::CLB5 cells is not downregulated in response to mating factor, cells arrest in the first cycle after addition of mating factor with a similar sensitivity as wild-type cells. However, whereas wild-type cells treated with mating factor arrest specifically in G1 phase as unbudded cells with unreplicated DNA (pre-START), most cln−GAL1::CLB5 cells arrest as budded post-START cells with replicated DNA. Our findings demonstrate the ability of post-START cells to arrest in response to mating factor and provide novel evidence for mechanisms that contribute to restrict mating factor-induced arrest in wild-type cells to the G1 phase of the cell cycle. Received: 25 September 1997 / Accepted: 9 November 1997     

Negative regulation of G1 and G2 by S-phase cyclins of Saccharomyces cerevisiae.

Cell cycle progression in the budding yeast Saccharomyces cerevisiae is controlled by the Cdc28 protein kinase, which is sequentially activated by different sets of cyclins. Previous genetic analysis has revealed that two B-type cyclins, Clb5 and Clb6, have a positive role in DNA replication. In the present study, we show, in addition, that these cyclins negatively regulate G1- and G2-specific functions. The consequences of this negative regulation were most apparent in clb6 mutants, which had a shorter pre-Start G1 phase as well as a shorter G2 phase than congenic wild-type cells. As a consequence, clb6 mutants grew and proliferated more rapidly than wild-type cells. It was more difficult to assess the role of Clb5 in G1 and G2 by genetic analysis because of the extreme prolongation of S phase in clb5 mutants. Nevertheless, both Clb5 and Clb6 were shown to be responsible for down-regulation of the protein kinase activities associated with Cln2, a G1 cyclin, and Clb2, a mitotic cyclin, in vivo. These observations are consistent with the observed cell cycle phase accelerations associated with the clb6 mutant and are suggestive of similar functions for Clb5. Genetic evidence suggested that the inhibition of mitotic cyclin-dependent kinase activities was dependent on and possibly mediated through the CDC6 gene product. Thus, Clb5 and Clb6 may stabilize S phase by promoting DNA replication while inhibiting other cell cycle activities.     

Identification of Clb2 residues required for Swe1 regulation of Clb2-Cdc28 in Saccharomyces cerevisiae

Wee1 kinases regulate the cell cycle through inhibitory phosphorylation of cyclin-dependent kinases (CDKs). Eukaryotic cells express multiple CDKs, each having a kinase subunit (Cdk) and a regulatory "cyclin" subunit that function at different stages of the cell cycle to regulate distinct processes. The cyclin imparts specificity to CDK-substrate interactions and also determines whether a particular CDK is subject to Wee1 regulation. Saccharomyces Wee1 (Swe1) inhibits Cdc28 (Cdk1) associated with the mitotic cyclin, Clb2, but not with the G(1) (Cln1, -2, and -3) or the S-phase (Clb5 and -6) cyclins. Here, we show that this specificity depends on two amino acids associated with a conserved "hydrophobic patch" (HP) motif on the cyclin surface, which mediates specificity of CDK-substrate interactions. Mutation of Clb2 residues N260 and K270 largely abrogates Clb2-Cdc28 regulation by Swe1, and reciprocal mutation of the corresponding residues in Clb5 can subject Clb5-Cdc28 to regulation by Swe1. Swe1 phosphorylation by Clb2-Cdc28, which is thought to activate Swe1 kinase, depends on N260 and K270, suggesting that specific regulation of Clb2-Cdc28 by Swe1 derives from the specific ability of Clb2 to target Swe1 for activating phosphorylation. The stable association of Swe1 with Clb2-Cdc28 also depends on these residues, suggesting that Swe1 may competitively inhibit Clb2-Cdc28 interactions with substrates, in addition to its well-known function as a regulator of CDK activity through tyrosine phosphorylation.     

CDK-Dependent Nuclear Localization of B-Cyclin Clb1 Promotes FEAR Activation during Meiosis I in Budding Yeast

Cyclin-dependent kinases (CDK) are master regulators of the cell cycle in eukaryotes. CDK activity is regulated by the presence, post-translational modification and spatial localization of its regulatory subunit cyclin. In budding yeast, the B-cyclin Clb1 is phosphorylated and localizes to the nucleus during meiosis I. However the functional significance of Clb1''s phosphorylation and nuclear localization and their mutual dependency is unknown. In this paper, we demonstrate that meiosis-specific phosphorylation of Clb1 requires its import to the nucleus but not vice versa. While Clb1 phosphorylation is dependent on activity of both CDK and polo-like kinase Cdc5, its nuclear localization requires CDK but not Cdc5 activity. Furthermore we show that increased nuclear localization of Clb1 during meiosis enhances activation of FEAR (Cdc Fourteen Early Anaphase Release) pathway. We discuss the significance of our results in relation to regulation of exit from meiosis I.     

Unraveling interactions of cell cycle-regulating proteins Sic1 and B-type cyclins in living yeast cells: a FLIM-FRET approach

Sic1, cyclin-dependent kinase inhibitor of budding yeast, is synthesized in anaphase and largely degraded at the S-phase onset to regulate timing of DNA synthesis. Sic1 interacts with phase-specific B-type cyclin (Clb)-kinase (Cdk1) complexes, central regulators in cell cycle control. Its appearance is timed to mediate reduction in kinase activities at appropriate stages. Clbs are unstable proteins with extremely short half-lives. Interactions of Sic1 with Clbs have been detected both in vitro and in vivo by high-throughput genome-wide screenings. Furthermore, we have recently shown that Sic1 regulates waves of Clbs, acting as a timer in their appearance, thus controlling Cdk1-Clbs activation. The molecular mechanism is not yet fully understood but is hypothesized to occur via stoichiometric binding of Sic1 to Cdk1-Clb complexes. Using F?rster resonance energy transfer (FRET) via fluorescence lifetime imaging microscopy (FLIM), we showed association of Sic1 to Clb cyclins in living yeast cells. This finding is consistent with the notion that inhibition of kinase activity can occur over the whole cell cycle progression despite variable Sic1 levels. Specifically, Sic1/Clb3 interaction was observed for the first time, and Sic1/Clb2 and Sic1/Clb5 pairs were confirmed, but no Sic1/Clb4 interaction was found, which suggests that, despite high functional homology between Clbs, only some of them can target Sic1 function in vivo.     

Cks1 is required for G(1) cyclin-cyclin-dependent kinase activity in budding yeast

p13(suc1) (Cks) proteins have been implicated in the regulation of cyclin-dependent kinase (CDK) activity. However, the mechanism by which Cks influences the function of cyclin-CDK complexes has remained elusive. We show here that Cks1 is required for the protein kinase activity of budding yeast G(1) cyclin-CDK complexes. Cln2 and Cdc28 subunits coexpressed in baculovirus-infected insect cells fail to exhibit protein kinase activity towards multiple substrates in the absence of Cks1. Cks1 can both stabilize Cln2-Cdc28 complexes and activate intact complexes in vitro, suggesting that it plays multiple roles in the biogenesis of active G(1) cyclin-CDK complexes. In contrast, Cdc28 forms stable, active complexes with the B-type cyclins Clb4 and Clb5 regardless of whether Cks1 is present. The levels of Cln2-Cdc28 and Cln3-Cdc28 protein kinase activity are severely reduced in cks1-38 cell extracts. Moreover, phosphorylation of G(1) cyclins, which depends on Cdc28 activity, is reduced in cks1-38 cells. The role of Cks1 in promoting G(1) cyclin-CDK protein kinase activity both in vitro and in vivo provides a simple molecular rationale for the essential role of CKS1 in progression through G(1) phase in budding yeast.     

Mouse cyclin-dependent kinase (Cdk) 5 is a functional homologue of a yeast Cdk, pho85 kinase

Mouse cyclin-dependent kinase (Cdk) 5 and yeast Pho85 kinase share similarities in structure as well as in the regulation of their activity. We found that mouse Cdk5 kinase produced in pho85Delta mutant cells could suppress some of pho85Delta mutant phenotypes including failure to grow on nonfermentable carbon sources, morphological defects, and growth defect caused by Pho4 or Clb2 overproduction. We also demonstrated that Cdk5 coimmunoprecipitated with Pho85-cyclins including Pcl1, Pcl2, Pcl6, Pcl9, and Pho80, and that the immunocomplex could phosphorylate Pho4, a native substrate of Pho85 kinase. Thus mouse Cdk5 is a functional homologue of yeast Pho85 kinase.     

The function and regulation of budding yeast Swe1 in response to interrupted DNA synthesis

Periodically regulated cyclin-dependent kinase (Cdk) is required for DNA synthesis and mitosis. Hydroxyurea (HU) inhibits DNA synthesis by depleting dNTPs, the basic unit for DNA synthesis. HU treatment triggers the S-phase checkpoint, which arrests cells at S-phase, inhibits late origin firing and stabilizes replication forks. Using budding yeast as a model system, we found that Swe1, a negative regulator of Cdk, appears at S-phase and accumulates in HU treatment cells. Interestingly, this accumulation is not dependent on S-phase checkpoint. Deltahsl1, Deltahsl7, and cdc5-2 mutants, which have defects in Swe1 degradation, show HU sensitivity because of high Swe1 protein levels. We further demonstrated that their HU sensitivity is not a result of DNA damage accumulation or incomplete DNA synthesis; instead the sensitivity is due to their dramatically delayed recovery from HU-induced S-phase arrest. Strikingly, our in vivo data indicate that Swe1 inhibits the kinase activity of Clb2-Cdk1, but not that of Clb5-Cdk1. Therefore, S-phase accumulated Swe1 prevents Clb2-Cdk1-mediated mitotic activities, but has little effects on Clb5-Cdk1-associated S-phase progression.     

The Ubp15 deubiquitinase promotes timely entry into S phase in Saccharomyces cerevisiae

The anaphase-promoting complex in partnership with its activator, Cdh1, is an E3 ubiquitin ligase responsible for targeting cell cycle proteins during G1 phase. In the budding yeast Saccharomyces cerevisiae, Cdh1 associates with the deubiquitinating enzyme Ubp15, but the significance of this interaction is unclear. To better understand the physiological role(s) of Ubp15, we examined cell cycle phenotypes of cells lacking Ubp15. We found that ubp15∆ cells exhibited delayed progression from G1 into S phase and increased sensitivity to the DNA synthesis inhibitor hydroxyurea. Both phenotypes of ubp15∆ cells were rescued by additional copies of the S-phase cyclin gene CLB5. Clb5 is an unstable protein targeted for proteasome-mediated degradation by several pathways. We found that during G1 phase, the APC^Cdh1-mediated degradation of Clb5 was accelerated in ubp15∆ cells. Ubp15 interacted with Clb5 independent of Cdh1 and deubiquitinated Clb5 in a reconstituted system. Thus deubiquitination by Ubp15 counteracts APC activity toward cyclin Clb5 to allow Clb5 accumulation and a timely entry into S phase.     

The cyclin-dependent kinase Cdc28p regulates distinct modes of Cdc6p proteolysis during the budding yeast cell cycle

BACKGROUND: Cdc28p, the major cyclin-dependent kinase in budding yeast, prevents re-replication within each cell cycle by preventing the reassembly of Cdc6p-dependent pre-replicative complexes (pre-RCs) once origins have fired. Cdc6p is a rapidly degraded protein that must be synthesised in each cell cycle and is present only during the G1 phase. RESULTS: We found that, at different times in the cell cycle, there are distinct modes of Cdc6p proteolysis. Before Start, Cdc6p proteolysis did not require either the anaphase-promoting complex (APC/C) or the SCF complex, which mediate the major cell cycle regulated ubiquitination pathways, nor did it require Cdc28p activity or any of the potential Cdc28p phosphorylation sites in Cdc6p. In fact, the activation of B cyclin (Clb)-Cdc28p kinase inactivated this pathway of Cdc6p degradation later in the cell cycle. Activation of the G1 cyclins (Clns) caused Cdc6p degradation to become extremely rapid. This degradation required the SCF(CDC4) and Cdc28p consensus sites in Cdc6p, but did not require Clb5 and Clb6. Later in the cell cycle, SCF(CDC4)-dependent Cdc6p proteolysis remained active but became less rapid. CONCLUSIONS: Levels of Cdc6p are regulated in several ways by the Cdc28p cyclin-dependent kinase. The Cln-dependent elimination of Cdc6p, which does not require the S-phase-promoting cyclins Clb5 and Clb6, suggests that the ability to assemble pre-RCs is lost before, not concomitant with, origin firing.     

The Saccharomyces cerevisiae Ptc1 protein phosphatase attenuates G2‐M cell cycle blockage caused by activation of the cell wall integrity pathway

Lack of the yeast Ptc1 Ser/Thr protein phosphatase results in numerous phenotypic defects. A parallel search for high‐copy number suppressors of three of these phenotypes (sensitivity to Calcofluor White, rapamycin and alkaline pH), allowed the isolation of 25 suppressor genes, which could be assigned to three main functional categories: maintenance of cell wall integrity (CWI), vacuolar function and protein sorting, and cell cycle regulation. The characterization of these genetic interactions strengthens the relevant role of Ptc1 in downregulating the Slt2‐mediated CWI pathway. We show that under stress conditions activating the CWI pathway the ptc1 mutant displays hyperphosphorylated Cdc28 kinase and that these cells accumulate with duplicated DNA content, indicative of a G2‐M arrest. Clb2‐associated Cdc28 activity was also reduced in ptc1 cells. These alterations are attenuated by mutation of the MKK1 gene, encoding a MAP kinase kinase upstream Slt2. Therefore, our data show that Ptc1 is required for proper G2‐M cell cycle transition after activation of the CWI pathway.     

Morphogenesis in the yeast cell cycle: regulation by Cdc28 and cyclins

Analysis of cell cycle regulation in the budding yeast Saccharomyces cerevisiae has shown that a central regulatory protein kinase, Cdc28, undergoes changes in activity through the cell cycle by associating with distinct groups of cyclins that accumulate at different times. The various cyclin/Cdc28 complexes control different aspects of cell cycle progression, including the commitment step known as START and mitosis. We found that altering the activity of Cdc28 had profound effects on morphogenesis during the yeast cell cycle. Our results suggest that activation of Cdc28 by G1 cyclins (Cln1, Cln2, or Cln3) in unbudded G1 cells triggers polarization of the cortical actin cytoskeleton to a specialized pre-bud site at one end of the cell, while activation of Cdc28 by mitotic cyclins (Clb1 or Clb2) in budded G2 cells causes depolarization of the cortical actin cytoskeleton and secretory apparatus. Inactivation of Cdc28 following cyclin destruction in mitosis triggers redistribution of cortical actin structures to the neck region for cytokinesis. In the case of pre-bud site assembly following START, we found that the actin rearrangement could be triggered by Cln/Cdc28 activation in the absence of de novo protein synthesis, suggesting that the kinase may directly phosphorylate substrates (such as actin-binding proteins) that regulate actin distribution in cells.     

Among B-type cyclins only CLB5 and CLB6 promote premeiotic S phase in Saccharomyces cerevisiae

The Saccharomyces cerevisiae cyclin Clb5 is required for premeiotic S phase, meiotic recombination, and successful progression through meiosis. Clb5 is not essential for mitotic proliferation because Clb1-Clb4 can support DNA replication in clb5 clb6 mutants. Clb1, Clb3, and Clb4 accumulate in clb5 clb6 cells during meiotic differentiation yet fail to promote premeiotic DNA replication. When expressed under the regulation of the CLB5 promoter, Clb1 and Clb3 accumulate and are active in the early stages of meiotic differentiation but cannot induce premeiotic DNA replication, suggesting that they do not target Cdk1 to the necessary substrates. The Clb5 hydrophobic patch (HP) residues are important for Clb5 function but this motif alone does not provide the specificity required for Clb5 to induce premeiotic S phase. Domain exchange experiments demonstrated that the amino terminus of Clb5 when fused to Clb3 confers upon Clb3 the ability to induce premeiotic S phase. Chimeric cyclins containing smaller regions of the Clb5 amino terminus displayed reduced ability to activate premeiotic DNA replication despite being more abundant and having greater associated histone H1 kinase activity than endogenous Clb5. These observations suggest that Clb5 has a unique ability to trigger premeiotic S phase and that the amino-terminal region of Clb5 contributes to its specificity and regulates the functions performed by the cyclin-Cdk complex.     

Differential function and expression of Saccharomyces cerevisiae B-type cyclins in mitosis and meiosis.

We have studied the patterns of expression of four B-type cyclins (Clbs), Clb1, Clb2, Clb3, and Clb4, and their ability to activate p34cdc28 during the mitotic and meiotic cell cycles of Saccharomyces cerevisiae. During the mitotic cell cycle, Clb3 and Clb4 were expressed and induced a kinase activity in association with p34cdc28 from early S phase up to mitosis. On the other hand, Clb1 and Clb2 were expressed and activated p34cdc28 later in the mitotic cell cycle, starting in late S phase and continuing up to mitosis. The pattern of expression of Clb3 and Clb4 suggests a possible role in the regulation of DNA replication as well as mitosis. Clb1 and Clb2, whose pattern of expression is similar to that of other known Clbs, are likely to have a role predominantly in the regulation of M phase. During the meiotic cell cycle, Clb1, Clb3, and Clb4 were expressed and induced a p34cdc28-associated kinase activity just before the first meiotic division. The fact that Clb3 and Clb4 were not synthesized earlier, in S phase, suggests that these cyclins, which probably have a role in S phase during the mitotic cell cycle, are not implicated in premeiotic S phase. Clb2, the primary mitotic cyclin in S. cerevisiae, was not detectable during meiosis. Sporulation experiments on strains deleted for one, two, or three Clbs indicate, in agreement with the biochemical data, that Clb1 is the primary cyclin for the regulation of meiosis, while Clb2 is not involved at all.     

Protein kinases involved in mating and osmotic stress in the yeast Kluyveromyces lactis

Systematic disruption of genes encoding kinases and mitogen-activated protein kinases (MAPKs) was performed in Kluyveromyces lactis haploid cells. The mutated strains were assayed by their capacity to mate and to respond to hyperosmotic stress. The K. lactis Ste11p (KlSte11p) MAPK kinase kinase (MAPKKK) was found to act in both mating and osmoresponse pathways while the scaffold KlSte5p and the MAPK KlFus3p appeared to be specific for mating. The p21-activated kinase KlSte20p and the kinase KlSte50p participated in both pathways. Protein association experiments showed interaction of KlSte50p and KlSte20p with Gα and Gβ, respectively, the G protein subunits involved in the mating pathway. Both KlSte50p and KlSte20p also showed interaction with KlSte11p. Disruption mutants of the K. lactis PBS2 (KlPBS2) and KlHOG1 genes of the canonical osmotic response pathway resulted in mutations sensitive to high salt and high sorbitol but dispensable for mating. Mutations that eliminate the MAPKK KlSte7p activity had a strong effect on mating and also showed sensitivity to osmotic stress. Finally, we found evidence of physical interaction between KlSte7p and KlHog1p, in addition to diminished Hog1p phosphorylation after a hyperosmotic shock in cells lacking KlSte7p. This study reveals novel roles for components of transduction systems in yeast.     

Dynamics of Cdk1 substrate specificity during the cell cycle

Cdk specificity is determined by the intrinsic selectivity of the active site and by substrate docking sites on the cyclin subunit. There is a long-standing debate about the relative importance of these factors in the timing of Cdk1 substrate phosphorylation. We analyzed major budding yeast cyclins (the G1/S-cyclin Cln2, S-cyclin Clb5, G2/M-cyclin Clb3, and M-cyclin Clb2) and found that the activity of Cdk1 toward the consensus motif increased gradually in the sequence Cln2-Clb5-Clb3-Clb2, in parallel with cell cycle progression. Further, we identified a docking element that compensates for the weak intrinsic specificity of Cln2 toward G1-specific targets. In addition, Cln2-Cdk1 showed distinct consensus site specificity, suggesting that cyclins do not merely activate Cdk1 but also modulate its active-site specificity. Finally, we identified several Cln2-, Clb3-, and Clb2-specific Cdk1 targets. We propose that robust timing and ordering of cell cycle events depend on gradual changes in the substrate specificity of Cdk1.     

APC/CCdh1 specific degradation of Hsl1 and Clb2 is required for proper stress responses of S. cerevisiae

Cdh1 activates the Anaphase Promoting Complex/Cyclosome (APC/CCdh1) throughout G1 to degrade key cell cycle proteins. Cdh1 is not essential for cell proliferation, in spite of the fact that overexpression of some its degradation substrates is highly toxic. We report here that cdh1Δ cells are sensitive to stresses that activate the CWI (Cell Wall Integrity) and Hog1 MAP kinase pathways. Stresses did not activate APC/CCdh1 and cellular sensitivity was thus clearly due to constitutively elevated substrate levels. To explore the contribution of stabilization of individual APC/CCdh1 substrates to stress sensitivity, we generated cell lines expressing stabilized substrate mutants under their endogenous promoters. Cells expressing stabilized Hsl1 were sensitive to caffeine and failed to activate the Slt2 pathway. Cells expressing partially stable Clb2 were particularly sensitive to different stresses, possibly due to reduced Sic1 levels. Cells expressing stabilized Cdc5 were much less stress sensitive. Interestingly sensitivity of cdh1Δ cells does not seem to be restricted to G1 but is manifested also during S and G2 when the APC/CCdh1 is inactive anyway. We thus hypothesize that a role of G1 specific APC/CCdh1 activity is to reset substrate levels to enables appropriate regulation of substrate accumulation in the subsequent phases of the cell cycle.