The emerging role of APC/CCdh1 in controlling differentiation, genomic stability and tumor suppression.

Deregulation of the G1/G0 phase of the cell cycle can lead to cancer. During G1, most cells commit alternatively to DNA replication and division, or to cell-cycle exit and differentiation. The anaphase-promoting complex or cyclosome (APC/C) activated by Cdh1 coordinately eliminates positive cell-cycle regulators as well as inhibitors of differentiation, thereby coupling cell-cycle exit and differentiation. Misregulation of Cdh1 thus has the potential to promote both cell-cycle re-entry and either perturbed differentiation or dedifferentiation. In addition, APC/C(Cdh1) is required to maintain genomic stability. As a result, loss of Cdh1 can contribute to tumorigenesis in the form of proliferation of poorly differentiated and genetically unstable cells.

Forced periodic expression of G1 cyclins phase-locks the budding yeast cell cycle.

Phase-locking (frequency entrainment) of an oscillator, in which a periodic extrinsic signal drives oscillations at a frequency different from the unperturbed frequency, is a useful property for study of oscillator stability and structure. The cell cycle is frequently described as a biochemical oscillator; however, because this oscillator is tied to key biological events such as DNA replication and segregation, and to cell growth (cell mass increase), it is unclear whether phase locking is possible for the cell cycle oscillator. We found that forced periodic expression of the G(1) cyclin CLN2 phase locks the cell cycle of budding yeast over a range of extrinsic periods in an exponentially growing monolayer culture. We characterize the behavior of cells in a pedigree using a return map to determine the efficiency of entrainment to the externally controlled pulse. We quantify differences between mothers and daughters and how synchronization of an expanding population differs from synchronization of a single oscillator. Mothers only lock intermittently whereas daughters lock completely and in a different period range than mothers. We can explain quantitative features of phase locking in both cell types with an analytically solvable model based on cell size control and how mass is partitioned between mother and daughter cells. A key prediction of this model is that size control can occur not only in G(1), but also later in the cell cycle under the appropriate conditions; this prediction is confirmed in our experimental data. Our results provide quantitative insight into how cell size is integrated with the cell cycle oscillator.

Retinoblastoma protein: combating algal bloom.

The discovery of a homolog of the retinoblastoma protein (Rb) in a single-celled eukaryote--the alga Chlamydomonas--promises new and surprising insights into Rb's function in cell-cycle regulation.

Mutations in SID2, a novel gene in Saccharomyces cerevisiae, cause synthetic lethality with sic1 deletion and may cause a defect during S phase.

SIC1 encodes a nonessential B-type cyclin/CDK inhibitor that functions at the G1/S transition and the exit from mitosis. To understand more completely the regulation of these transitions, mutations causing synthetic lethality with sic1 Delta were isolated. In this screen, we identified a novel gene, SID2, which encodes an essential protein that appears to be required for DNA replication or repair. sid2-1 sic1 Delta strains and sid2-21 temperature-sensitive strains arrest preanaphase as large-budded cells with a single nucleus, a short spindle, and an approximately 2C DNA content. RAD9, which is necessary for the DNA damage checkpoint, is required for the preanaphase arrest of sid2-1 sic1 Delta cells. Analysis of chromosomes in mutant sid2-21 cells by field inversion gel electrophoresis suggests the presence of replication forks and bubbles at the arrest. Deleting the two S phase cyclins, CLB5 and CLB6, substantially suppresses the sid2-1 sic1 Delta inviability, while stabilizing Clb5 protein exacerbates the defects of sid2-1 sic1 Delta cells. In synchronized sid2-1 mutant strains, the onset of replication appears normal, but completion of DNA synthesis is delayed. sid2-1 mutants are sensitive to hydroxyurea indicating that sid2-1 cells may suffer DNA damage that, when combined with additional insult, leads to a decrease in viability. Consistent with this hypothesis, sid2-1 rad9 cells are dead or very slow growing even when SIC1 is expressed.

Mechanisms controlling subcellular localization of the G(1) cyclins Cln2p and Cln3p in budding yeast.

Different G(1) cyclins confer functional specificity to the cyclin-dependent kinase (Cdk) Cdc28p in budding yeast. The Cln3p G(1) cyclin is localized primarily to the nucleus, while Cln2p is localized primarily to the cytoplasm. Both binding to Cdc28p and Cdc28p-dependent phosphorylation in the C-terminal region of Cln2p are independently required for efficient nuclear depletion of Cln2p, suggesting that this process may be physiologically regulated. The accumulation of hypophosphorylated Cln2 in the nucleus is an energy-dependent process, but may not involve the RAN GTPase. Phosphorylation of Cln2p is inefficient in small newborn cells obtained by elutriation, and this lowered phosphorylation correlates with reduced Cln2p nuclear depletion in newborn cells. Thus, Cln2p may have a brief period of nuclear residence early in the cell cycle. In contrast, the nuclear localization pattern of Cln3p is not influenced by Cdk activity. Cln3p localization requires a bipartite nuclear localization signal (NLS) located at the C terminus of the protein. This sequence is required for nuclear localization of Cln3p and is sufficient to confer nuclear localization to green fluorescent protein in a RAN-dependent manner. Mislocalized Cln3p, lacking the NLS, is much less active in genetic assays specific for Cln3p, but more active in assays normally specific for Cln2p, consistent with the idea that Cln3p localization explains a significant part of Clnp functional specificity.

Cyclin specificity: how many wheels do you need on a unicycle?

Cyclin-dependent kinase (CDK) activity is essential for eukaryotic cell cycle events. Multiple cyclins activate CDKs in all eukaryotes, but it is unclear whether multiple cyclins are really required for cell cycle progression. It has been argued that cyclins may predominantly act as simple enzymatic activators of CDKs; in opposition to this idea, it has been argued that cyclins might target the activated CDK to particular substrates or inhibitors. Such targeting might occur through a combination of factors, including temporal expression, protein associations, and subcellular localization.

Conservation and function of a potential substrate-binding domain in the yeast Clb5 B-type cyclin.

Cyclin A contains a region implicated in binding to the p27 inhibitor and to substrates. There is strong evolutionary conservation of surface residues contributing to this region in many cyclins, including yeast B-type cyclins, despite the absence of a yeast p27 homolog. The yeast S-phase B-type cyclin Clb5p interacted with mammalian p27 in a two-hybrid assay. This interaction was disrupted by mutations designed to disrupt hydrophobic interactions (hpm mutation) or hydrogen bonding (Q241A mutation) based on the cyclin A-p27 crystal structure. In contrast, mutation of the Clb5p p27-binding domain only slightly reduced binding and inhibition by the Sic1p Clb-Cdc28p kinase inhibitor. Mutations disrupting the p27-binding domain strongly reduced Clb5p biological activity in diverse assays without reducing Clb5p-associated kinase activity. An analogous hpm mutation in the mitotic cyclin Clb2p reduced mitotic function, but in some assays this mutation increased the ability of Clb2p to perform functions normally restricted to Clb5p. These results support the idea of a modular, structurally conserved cyclin domain involved in substrate targeting.

Testing cyclin specificity in the exit from mitosis.

Cyclical inactivation of B-type cyclins has been proposed to be required for alternating DNA replication and mitosis. Destruction box-dependent Clb5p degradation is strongly increased in mitotic cells, and constitutive overexpression of Clb5p lacking the destruction box resulted in rapid accumulation of inviable cells, frequently multiply budded, with DNA contents ranging from unreplicated to apparently fully replicated. Loss of viability correlated with retention of nuclear Clb5p at the time of nuclear division. CLB2-Deltadb overexpression that was quantitatively comparable to CLB5-Deltadb overexpression with respect to Clb protein production and Clb-associated kinase activity resulted in a distinct phenotype: reversible mitotic arrest with uniformly replicated DNA. Simultaneous overexpression of CLB2-Deltadb and CLB5-Deltadb overexpressers similarly resulted in a uniform arrest with replicated DNA, and this arrest was significantly more reversible than that observed with CLB5-Deltadb overexpression alone. These results suggest that Clb2p and not Clb5p can efficiently block mitotic completion. We speculate that CLB5-Deltadb overexpression may be lethal, because persistence of high nuclear Clb5p-associated kinase throughout mitosis leads to failure to load origins of replication, thus preventing DNA replication in the succeeding cell cycle.

Genetic analysis of the relationship between activation loop phosphorylation and cyclin binding in the activation of the Saccharomyces cerevisiae Cdc28p cyclin-dependent kinase.

We showed recently that a screen for mutant CDC28 with improved binding to a defective Cln2p G1 cyclin yielded a spectrum of mutations similar to those yielded by a screen for intragenic suppressors of the requirement for activation loop phosphorylation (T169E suppressors). Recombination among these mutations yielded CDC28 mutants that bypassed the G1 cyclin requirement. Here we analyze further the interrelationship between T169E suppression, interaction with defective cyclin, and G1 cyclin bypass. DNA shuffling of mutations from the various screens and recombination onto a T169E-encoding 3' end yielded CDC28 mutants with strong T169E suppression. Some of the strongest T169E suppressors could suppress the defective Cln2p G1 cyclin even while retaining T169E. The strong T169E suppressors did not exhibit bypass of the G1 cyclin requirement but did so when T169E was reverted to T. These results suggested that for these mutants, activation loop phosphorylation and cyclin binding might be alternative means of activation rather than independent requirements for activation (as with wild type). These results suggest mechanistic overlap between the conformational shift induced by cyclin binding and that induced by activation loop phosphorylation. This conclusion was supported by analysis of suppressors of a mutation in the Cdk phosphothreonine-binding pocket created by cyclin binding.

Mutations in CDC14 result in high sensitivity to cyclin gene dosage in Saccharomyces cerevisiae.

We screened for mutations that resulted in lethality when the G1 cyclin Cln2p was overexpressed throughout the cell cycle in Saccharomyces cerevisiae. Mutations in five complementation groups were found to give this phenotype, and three of the mutated genes were identified as MEC1, NUP170, and CDC14. Mutations in CDC14 may have been recovered in the screen because Cdc14p may reduce the cyclin B (Clb)-associated Cdc28 kinase activity in late mitosis, and Cln2p may normally activate Clb-Cdc28 kinase activity by related mechanisms. In agreement with the idea that cdc14 mutations elevate Clb-Cdc28 kinase activity, deletion of the gene for the Clb-Cdc28 inhibitor Sic1 caused synthetic lethality with cdc14-1, as did the deletion of HCT1, which is required for proteolysis of Clb2p. Surprisingly, deletion of the gene for the major B-type cyclin, CLB2, also caused synthetic lethality with the cdc14-1 mutation. The clb2 cdc14 strains arrested with replicated but unseparated DNA and unseparated spindle pole bodies; this phenotype is distinct from the late mitotic arrest of the sic1::TRP1 cdc14-1 and the cdc14-1 hct1::LEU2 double mutants and of the cdc14 CLN2 overexpressor. We found genetic interactions between CDC14 and the replication initiator gene CDC6, extending previous observations of interactions between the late mitotic function of Cdc14p and control of DNA replication. We also describe genetic interactions between CDC28 and CDC14.

Distinct subcellular localization patterns contribute to functional specificity of the Cln2 and Cln3 cyclins of Saccharomyces cerevisiae.

The G(1) cyclins of budding yeast drive cell cycle initiation by different mechanisms, but the molecular basis of their specificity is unknown. Here we test the hypothesis that the functional specificity of G(1) cyclins is due to differential subcellular localization. As shown by indirect immunofluorescence and biochemical fractionation, Cln3p localization appears to be primarily nuclear, with the most obvious accumulation of Cln3p to the nuclei of large budded cells. In contrast, Cln2p localizes to the cytoplasm. We were able to shift localization patterns of truncated Cln3p by the addition of nuclear localization and nuclear export signals, and we found that nuclear localization drives a Cln3p-like functional profile, while cytoplasmic localization leads to a partial shift to a Cln2p-like functional profile. Therefore, forcing Cln3p into a Cln2p-like cytoplasmic localization pattern partially alters the functional specificity of Cln3p toward that of Cln2p. These results suggest that there are CLN-dependent cytoplasmic and nuclear events important for cell cycle initiation. This is the first indication of a cytoplasmic function for a cyclin-dependent kinase. The data presented here support the idea that cyclin function is regulated at the level of subcellular localization and that subcellular localization contributes to the functional specificity of Cln2p and Cln3p.

Directed evolution to bypass cyclin requirements for the Cdc28p cyclin-dependent kinase.

To identify cyclin-dependent kinase mutants with relaxed cyclin requirements, CDC28 alleles were selected that could rescue a yeast strain expressing as its only CLN G1 cyclin a mutant Cln2p (K129A,E183A) that is defective for Cdc28p binding. Rescue of this strain by mutant CDC28 was dependent upon the mutant cln2-KAEA, but additional mutagenesis and DNA shuffling yielded multiply mutant CDC28-BYC alleles (bypass of CLNs) that could support highly efficient cell cycle initiation in the complete absence of CLN genes. By gel filtration chromatography, one of the mutant Cdc28 proteins exhibited kinase activity associated with cyclin-free monomer. Thus, the mutants' CLN bypass activity might result from constitutive, cyclin-independent activity, suggesting that Cdk targeting by cyclins is not required for cell cycle initiation.

Specialization and targeting of B-type cyclins.

The B-type cyclins of S. cerevisiae are diversified with respect to time of expression during the cell cycle as well as biological function. We replaced the early-expressed CLB5 coding sequence with the late-expressed CLB2 coding sequence, at the CLB5 locus. CLB5::CLB2 exhibited almost no rescue of clb5-specific replication defects, although it could rescue clb1 clb2 lethality, and in synchronized cells Clb2p-associated kinase activity from CLB5::CLB2 rose early in the cell cycle, similar to that of Clb5p. Mutagenesis of a potential substrate-targeting domain of CLB5 reduced biological activity without reducing Clb5p-associated kinase activity. Thus, Clb5p may have targeting domains required for CLB5-specific biological activity.

Accurate quantitation of protein expression and site-specific phosphorylation.

A mass spectrometry-based method is described for simultaneous identification and quantitation of individual proteins and for determining changes in the levels of modifications at specific sites on individual proteins. Accurate quantitation is achieved through the use of whole-cell stable isotope labeling. This approach was applied to the detection of abundance differences of proteins present in wild-type versus mutant cell populations and to the identification of in vivo phosphorylation sites in the PAK-related yeast Ste20 protein kinase that depend specifically on the G1 cyclin Cln2. The present method is general and affords a quantitative description of cellular differences at the level of protein expression and modification, thus providing information that is critical to the understanding of complex biological phenomena.

Interaction between the MEC1-dependent DNA synthesis checkpoint and G1 cyclin function in Saccharomyces cerevisiae.

The completion of DNA synthesis in yeast is monitored by a checkpoint that requires MEC1 and RAD53. Here we show that deletion of the Saccharomyces cerevisiae G1 cyclins CLN1 and CLN2 suppressed the essential requirement for MEC1 function. Wild-type levels of CLN1 and CLN2, or overexpression of CLN1, CLN2, or CLB5, but not CLN3, killed mec1 strains. We identified RNR1, which encodes a subunit of ribonucleotide reductase, as a high-copy suppressor of the lethality of mec1 GAL1-CLN1. Northern analysis demonstrated that RNR1 expression is reduced by CLN1 or CLN2 overexpression. Because limiting RNR1 expression would be expected to decrease dNTP pools, CLN1 and CLN2 may cause lethality in mec1 strains by causing initiation of DNA replication with inadequate dNTPs. In contrast to mec1 mutants, MEC1 strains with low dNTPs would be able to delay S phase and thereby remain viable. We propose that the essential function for MEC1 may be the same as its checkpoint function during hydroxyurea treatment, namely, to slow S phase when nucleotides are limiting. In a cln1 cln2 background, a prolonged period of expression of genes turned on at the G1-S border, such as RNR1, has been observed. Thus deletion of CLN1 and CLN2 could function similarly to overexpression of RNR1 in suppressing mec1 lethality.

Potential regulation of Ste20 function by the Cln1-Cdc28 and Cln2-Cdc28 cyclin-dependent protein kinases.

The activity of the Saccharomyces cerevisiae pheromone signal transduction pathway is regulated by Cln1/2-Cdc28 cyclin-dependent kinase. High level expression of CLN2 can repress activation of the pathway by mating factor or by deletion of the alpha-subunit of the heterotrimeric G-protein. We now show that CLN2 overexpression can also repress FUS1 induction if the signaling pathway is activated at the level of the beta-subunit of the G-protein (STE4) but not when activated at the level of downstream kinases (STE20 and STE11) or at the level of the transcription factor STE12. This epistatic analysis indicates that repression of pheromone signaling pathway by Cln2-Cdc28 kinase takes place at a level around STE20. In agreement with this, a marked reduction in the electrophoretic mobility of the Ste20 protein is observed at the time in the cell cycle of maximal expression of CLN2. This mobility change is constitutive in cells overexpressing CLN2 and absent in cells lacking CLN1 and CLN2. These changes in electrophoretic mobility correlate with repression of pheromone signaling and suggest Ste20 as a target for repression of signaling by G1 cyclins. Two morphogenic pathways for which Ste20 is essential, pseudohyphal differentiation and haploid-invasive growth, also require CLN1 and CLN2. Together with the previous observation that Cln1 and Cln2 are required for the function of Ste20 in cytokinesis, this suggests that Cln1 and Cln2 regulate the biological activity of Ste20 by promoting morphogenic functions, while inhibiting the mating factor signal transduction function.

CLB5-dependent activation of late replication origins in S. cerevisiae.

Replication origins in chromosomes are activated at specific times during the S phase. We show that the B-type cyclins are required for proper execution of this temporal program. clb5 cells activate early origins but not late origins, explaining the previously described long clb5 S phase. Origin firing appears normal in cIb6 mutants. In clb5 clb6 double mutant cells, the late origin firing defect is suppressed, accounting for the normal duration of the phase despite its delayed onset. Therefore, Clb5p promotes the timely activation of early and late origins, but Clb6p can activate only early origins. In clb5 clb6 mutants, the other B-type cyclins (Clb1-4p) promote an S phase during which both early and late replication origins fire.

The mating factor response pathway regulates transcription of TEC1, a gene involved in pseudohyphal differentiation of Saccharomyces cerevisiae.

The transcription factor Tec1 is involved in pseudohyphal differentiation and agar-invasive growth of Saccharomyces cerevisiae cells. The sole element in the TEC1 promoter that has thus far been shown to control Tec1 function is the filament response element. We find that the TEC1 promoter also contains several pheromone response element sequences which are likely to be functional: TEC1 transcription is induced by mating factor, cell cycle regulated and dependent on the Ste4, Ste18 and Ste5 components of the mating factor signal transduction pathway. Using alleles of the transcription factor Ste12 that are defective in DNA binding, transcriptional induction or cooperativity with other transcription factors, we find little correlation between TEC1 transcript levels and agar-invasive growth.

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.

Pheromone-dependent G1 cell cycle arrest requires Far1 phosphorylation, but may not involve inhibition of Cdc28-Cln2 kinase, in vivo.

In yeast, the pheromone alpha-factor acts as an antiproliferative factor that induces G1 arrest and cellular differentiation. Previous data have indicated that Far1, a factor dedicated to pheromone-induced cell cycle arrest, is under positive and negative posttranslational regulation. Phosphorylation by the pheromone-stimulated mitogen-activated protein (MAP) kinase Fus3 has been thought to enhance the binding of Far1 to G1-specific cyclin-dependent kinase (Cdk) complexes, thereby inhibiting their catalytic activity. Cdk-dependent phosphorylation events were invoked to account for the high instability of Far1 outside early G1 phase. To confirm any functional role of Far1 phosphorylation, we undertook a systematic mutational analysis of potential MAP kinase and Cdk recognition motifs. Two putative phosphorylation sites that strongly affect Far1 behavior were identified. A change of serine 87 to alanine prevents the cell cycle-dependent degradation of Far1, causing enhanced sensitivity to pheromone. In contrast, threonine 306 seems to be an important recipient of an activating modification, as substitutions at this position abolish the G1 arrest function of Far1. Only the phosphorylated wild-type Far1 protein, not the T306-to-A substitution product, can be found in stable association with the Cdc28-Cln2 complex. Surprisingly, Far1-associated Cdc28-Cln2 complexes are at best moderately inhibited in immunoprecipitation kinase assays, suggesting unconventional inhibitory mechanisms of Far1.