Analog-sensitive Cdk1 as a tool to study mitotic exit: protein phosphatase 1 is required downstream from Cdk1 inactivation in budding yeast.

We show that specific inactivation of the protein kinase Cdk1/cyclin B (Cdc28/Clb2) triggers exit from mitosis in the budding yeast Saccharomyces cerevisiae. Cells carrying the allele cdc28-as1, which makes Cdk1 (Cdc28) uniquely sensitive to the ATP analog 1NM-PP1, were arrested with spindle poisons and then treated with 1NM-PP1 to inhibit Cdk1. This caused the cells to leave mitosis and enter G1-phase as shown by initiation of rebudding (without cytokinesis), induction of mating projections ("shmoos") by α-factor, stabilization of Sic1, and degradation of Clb2. It is known that Cdk1 must be inactivated for cells to exit mitosis, but our results show that inactivation of Cdk1 is not only necessary but also sufficient to initiate the transition from mitosis to G1-phase. This result suggests a system in which to test requirements for particular gene products downstream from Cdk1 inactivation, for example, by combining cdc28-as1 with conditional mutations in the genes of interest. Using this approach, we demonstrate that protein phosphatase 1 (PPase1; Glc7 in S. cerevisiae) is required for mitotic exit and reestablishment of interphase following Cdk1 inactivation. This system could be used to test the need for other protein phosphatases downstream from Cdk1 inactivation, such as PPase 2A and Cdc14, and it could be combined with phosphoproteomics to gain information about the substrates that the various phosphatases act upon during mitotic exit.

Mitochondrial respiration promotes Cdc37-dependent stability of the Cdk1 homolog Cdc28.

Cdc28, the homolog of mammalian Cdk1, is a conserved key regulatory kinase for all major cell cycle transitions in yeast. We have found that defects in mitochondrial respiration (including deletion of ATP2, an ATP synthase subunit) inhibit growth of cells carrying a degron allele of Cdc28 (cdc28td) or Cdc28 temperature-sensitive mutations (cdc28-1 and cdc28-1N) at semi-permissive temperatures. Loss of cell proliferation in the atp2Δcdc28td double mutant is associated with aggravated cell cycle arrest and mitochondrial dysfunction, including mitochondrial hyperpolarization and fragmentation. Unexpectedly, in mutants defective in mitochondrial respiration, steady-state protein levels of mutant cdc28 are strongly reduced, accounting for the aggravated growth defects. Stability of Cdc28 is promoted by the Hsp90-Cdc37 chaperone complex. Our results show that atp2Δcdc28td double-mutant cells, but not single mutants, are sensitive to chemical inhibition of the Hsp90-Cdc37 complex, and exhibit reduced levels of additional Hsp90-Cdc37 client kinases, suggesting an inhibition of this complex. In agreement, overexpression of CDC37 improved atp2Δcdc28td cell growth and Cdc28 levels. Overall, our study shows that simultaneous disturbance of mitochondrial respiration and Cdc28 activity reduces the capacity of Cdc37 to chaperone client kinases, leading to growth arrest.

G1 cyclin-Cdk promotes cell cycle entry through localized phosphorylation of RNA polymerase II.

Cell division is thought to be initiated by cyclin-dependent kinases (Cdks) inactivating key transcriptional inhibitors. In budding yeast, the G1 cyclin Cln3-Cdk1 complex is thought to directly phosphorylate the Whi5 protein, thereby releasing the transcription factor SBF and committing cells to division. We report that Whi5 is a poor substrate of Cln3-Cdk1, which instead phosphorylates the RNA polymerase II subunit Rpb1's C-terminal domain on S5 of its heptapeptide repeats. Cln3-Cdk1 binds SBF-regulated promoters and Cln3's function can be performed by the canonical S5 kinase Ccl1-Kin28 when synthetically recruited to SBF. Thus, we propose that Cln3-Cdk1 triggers cell division by phosphorylating Rpb1 at SBF-regulated promoters to promote transcription. Our findings blur the distinction between cell cycle and transcriptional Cdks to highlight the ancient relationship between these two processes.

Cdc42-Specific GTPase-Activating Protein Rga1 Squelches Crosstalk between the High-Osmolarity Glycerol (HOG) and Mating Pheromone Response MAPK Pathways.

Eukaryotes utilize distinct mitogen/messenger-activated protein kinase (MAPK) pathways to evoke appropriate responses when confronted with different stimuli. In yeast, hyperosmotic stress activates MAPK Hog1, whereas mating pheromones activate MAPK Fus3 (and MAPK Kss1). Because these pathways share several upstream components, including the small guanosine-5'-triphosphate phosphohydrolase (GTPase) cell-division-cycle-42 (Cdc42), mechanisms must exist to prevent inadvertent cross-pathway activation. Hog1 activity is required to prevent crosstalk to Fus3 and Kss1. To identify other factors required to maintain signaling fidelity during hypertonic stress, we devised an unbiased genetic selection for mutants unable to prevent such crosstalk even when active Hog1 is present. We repeatedly isolated truncated alleles of RGA1, a Cdc42-specific GTPase-activating protein (GAP), each lacking its C-terminal catalytic domain, that permit activation of the mating MAPKs under hyperosmotic conditions despite Hog1 being present. We show that Rga1 down-regulates Cdc42 within the high-osmolarity glycerol (HOG) pathway, but not the mating pathway. Because induction of mating pathway output via crosstalk from the HOG pathway takes significantly longer than induction of HOG pathway output, our findings suggest that, under normal conditions, Rga1 contributes to signal insulation by limiting availability of the GTP-bound Cdc42 pool generated by hypertonic stress. Thus, Rga1 action contributes to squelching crosstalk by imposing a type of "kinetic proofreading". Although Rga1 is a Hog1 substrate in vitro, we eliminated the possibility that its direct Hog1-mediated phosphorylation is necessary for its function in vivo. Instead, we found first that, like its paralog Rga2, Rga1 is subject to inhibitory phosphorylation by the S. cerevisiae cyclin-dependent protein kinase 1 (Cdk1) ortholog Cdc28 and that hyperosmotic shock stimulates its dephosphorylation and thus Rga1 activation. Second, we found that Hog1 promotes Rga1 activation by blocking its Cdk1-mediated phosphorylation, thereby allowing its phosphoprotein phosphatase 2A (PP2A)-mediated dephosphorylation. These findings shed light on why Hog1 activity is required to prevent crosstalk from the HOG pathway to the mating pheromone response pathway.

Compensation for the absence of the catalytically active half of DNA polymerase ε in yeast by positively selected mutations in CDC28.

Current eukaryotic replication models postulate that leading and lagging DNA strands are replicated predominantly by dedicated DNA polymerases. The catalytic subunit of the leading strand DNA polymerase ε, Pol2, consists of two halves made of two different ancestral B-family DNA polymerases. Counterintuitively, the catalytically active N-terminal half is dispensable, while the inactive C-terminal part is required for viability. Despite extensive studies of yeast Saccharomyces cerevisiae strains lacking the active N-terminal half, it is still unclear how these strains survive and recover. We designed a robust method for constructing mutants with only the C-terminal part of Pol2. Strains without the active polymerase part show severe growth defects, sensitivity to replication inhibitors, chromosomal instability, and elevated spontaneous mutagenesis. Intriguingly, the slow-growing mutant strains rapidly accumulate fast-growing clones. Analysis of genomic DNA sequences of these clones revealed that the adaptation to the loss of the catalytic N-terminal part of Pol2 occurs by a positive selection of mutants with improved growth. Elevated mutation rates help generate sufficient numbers of these variants. Single nucleotide changes in the cell cycle-dependent kinase gene, CDC28, improve the growth of strains lacking the N-terminal part of Pol2, and rescue their sensitivity to replication inhibitors and, in parallel, lower mutation rates. Our study predicts that changes in mammalian homologs of cyclin-dependent kinases may contribute to cellular responses to the leading strand polymerase defects.

Post-translational Regulation of DNA Polymerase η, a Connection to Damage-Induced Cohesion in Saccharomyces cerevisiae.

Double-strand breaks that are induced postreplication trigger establishment of damage-induced cohesion in Saccharomyces cerevisiae, locally at the break site and genome-wide on undamaged chromosomes. The translesion synthesis polymerase, polymerase η, is required for generation of damage-induced cohesion genome-wide. However, its precise role and regulation in this process is unclear. Here, we investigated the possibility that the cyclin-dependent kinase Cdc28 and the acetyltransferase Eco1 modulate polymerase η activity. Through in vitro phosphorylation and structure modeling, we showed that polymerase η is an attractive substrate for Cdc28 Mutation of the putative Cdc28-phosphorylation site Ser14 to Ala not only affected polymerase η protein level, but also prevented generation of damage-induced cohesion in vivo We also demonstrated that Eco1 acetylated polymerase η in vitro Certain nonacetylatable polymerase η mutants showed reduced protein level, deficient nuclear accumulation, and increased ultraviolet irradiation sensitivity. In addition, we found that both Eco1 and subunits of the cohesin network are required for cell survival after ultraviolet irradiation. Our findings support functionally important Cdc28-mediated phosphorylation, as well as post-translational modifications of multiple lysine residues that modulate polymerase η activity, and provide new insights into understanding the regulation of polymerase η for damage-induced cohesion.

Cdk1 gates cell cycle-dependent tRNA synthesis by regulating RNA polymerase III activity.

tRNA genes are transcribed by RNA polymerase III (RNAPIII). During recent years it has become clear that RNAPIII activity is strictly regulated by the cell in response to environmental cues and the homeostatic status of the cell. However, the molecular mechanisms that control RNAPIII activity to regulate the amplitude of tDNA transcription in normally cycling cells are not well understood. Here, we show that tRNA levels fluctuate during the cell cycle and reveal an underlying molecular mechanism. The cyclin Clb5 recruits the cyclin dependent kinase Cdk1 to tRNA genes to boost tDNA transcription during late S phase. At tDNA genes, Cdk1 promotes the recruitment of TFIIIC, stimulates the interaction between TFIIIB and TFIIIC, and increases the dynamics of RNA polymerase III in vivo. Furthermore, we identified Bdp1 as a putative Cdk1 substrate in this process. Preventing Bdp1 phosphorylation prevented cell cycle-dependent recruitment of TFIIIC and abolished the cell cycle-dependent increase in tDNA transcription. Our findings demonstrate that under optimal growth conditions Cdk1 gates tRNA synthesis in S phase by regulating the RNAPIII machinery, revealing a direct link between the cell cycle and RNAPIII activity.

Swe1 and Mih1 regulate mitotic spindle dynamics in budding yeast via Bik1.

The mitotic spindle is a very dynamic structure that is built de novo and destroyed at each round of cell division. In order to perform its fundamental function during chromosome segregation, mitotic spindle dynamics must be tightly coordinated with other cell cycle events. These changes are driven by several protein kinases, phosphatases and microtubule-associated proteins. In budding yeast, the kinase Swe1 and the phosphatase Mih1 act in concert in controlling the phosphorylation state of Cdc28, the catalytic subunit of Cdk1, the major regulator of the cell cycle. In this study we show that Swe1 and Mih1 are also involved in the control of mitotic spindle dynamics. Our data indicate that Swe1 and the Polo-like kinase Cdc5 control the balance between phosphorylated and unphosphorylated forms of Mih1, which is, in turn, important for mitotic spindle elongation. Moreover, we show that the microtubule-associated protein Bik1 is a phosphoprotein, and that Swe1 and Mih1 are both involved in controlling phosphorylation of Bik1. These results uncover new players and provide insights into the complex regulation of mitotic spindle dynamics.

Structural changes induced by L50P and I61T single mutations of ubiquitin affect cell cycle progression while impairing its regulatory and degradative functions in Saccharomyces cerevisiae.

Posttranslational conjugation of ubiquitin to proteins either regulates their function directly or concentration through ubiquitination dependent degradation. High degree of conservation of ubiquitin's sequence implies structural and functional importance of the conserved residues. Ubiquitin gene of Saccharomyces cerevisiae was evolved in vitro by us to study the significance of conserved residues. Present study investigates the structural changes in the protein resulting from the single mutations UbS20F, UbA46S, UbL50P, UbI61T and their functional consequences in the SUB60 strain of S. cerevisiae. Expression of UbL50P and UbI61T decreased Cdc28 protein kinase, enhanced Fus3 levels, caused dosage dependent lethality and at sublethal level produced drastic effects on stress tolerance, protein sorting, protein degradation by ubiquitin fusion degradation pathway and by lysosomes. UbS20F and UbA46S produced insignificant effects over the cells. All four mutations of ubiquitin were incorporated into polyubiquitin. However, polyubiquitination with K63 linkage decreased significantly in cells expressing UbL50P and UbI61T. Structural studies on UbL50P and UbI61T revealed distorted structure with greatly reduced α-helical and elevated ß-sheet contents, while UbS20F and UbA46S show mild structural alterations. Our results on functional efficacy of ubiquitin in relation to structural integrity may be useful for designing inhibitors to investigate and modulate eukaryotic cellular dynamics.

Legionella pneumophila prevents proliferation of its natural host Acanthamoeba castellanii.

Legionella pneumophila is a ubiquitous, pathogenic, Gram-negative bacterium responsible for legionellosis. Like many other amoeba-resistant microorganisms, L. pneumophila resists host clearance and multiplies inside the cell. Through its Dot/Icm type IV secretion system, the bacterium injects more than three hundred effectors that modulate host cell physiology in order to promote its own intracellular replication. Here we report that L. pneumophila prevents proliferation of its natural host Acanthamoeba castellanii. Infected amoebae could not undergo DNA replication and no cell division was observed. The Dot/Icm secretion system was necessary for L. pneumophila to prevent the eukaryotic proliferation. The absence of proliferation was associated with altered amoebal morphology and with a decrease of mRNA transcript levels of CDC2b, a putative regulator of the A. castellanii cell cycle. Complementation of CDC28-deficient Saccharomyces cerevisiae by the CDC2b cDNA was sufficient to restore proliferation of CDC28-deficient S. cerevisiae and suggests for the first time that CDC2b from A. castellanii could be functional and a bona fide cyclin-dependent kinase. Hence, our results reveal that L. pneumophila impairs proliferation of A. castellanii and this effect could involve the cell cycle protein CDC2b.

A Synthetic Dosage Lethal Genetic Interaction Between CKS1B and PLK1 Is Conserved in Yeast and Human Cancer Cells.

The CKS1B gene located on chromosome 1q21 is frequently amplified in breast, lung, and liver cancers. CKS1B codes for a conserved regulatory subunit of cyclin-CDK complexes that function at multiple stages of cell cycle progression. We used a high throughput screening protocol to mimic cancer-related overexpression in a library of Saccharomyces cerevisiae mutants to identify genes whose functions become essential only when CKS1 is overexpressed, a synthetic dosage lethal (SDL) interaction. Mutations in multiple genes affecting mitotic entry and mitotic exit are highly enriched in the set of SDL interactions. The interactions between Cks1 and the mitotic entry checkpoint genes require the inhibitory activity of Swe1 on the yeast cyclin-dependent kinase (CDK), Cdc28. In addition, the SDL interactions of overexpressed CKS1 with mutations in the mitotic exit network are suppressed by modulating expression of the CDK inhibitor Sic1. Mutation of the polo-like kinase Cdc5, which functions in both the mitotic entry and mitotic exit pathways, is lethal in combination with overexpressed CKS1 Therefore we investigated the effect of targeting the human Cdc5 ortholog, PLK1, in breast cancers with various expression levels of human CKS1B Growth inhibition by PLK1 knockdown correlates with increased CKS1B expression in published tumor cell data sets, and this correlation was confirmed using shRNAs against PLK1 in tumor cell lines. In addition, we overexpressed CKS1B in multiple cell lines and found increased sensitivity to PLK1 knockdown and PLK1 drug inhibition. Finally, combined inhibition of WEE1 and PLK1 results in less apoptosis than predicted based on an additive model of the individual inhibitors, showing an epistatic interaction and confirming a prediction of the yeast data. Thus, identification of a yeast SDL interaction uncovers conserved genetic interactions that can affect human cancer cell viability.

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.

The Yeast Cyclin-Dependent Kinase Routes Carbon Fluxes to Fuel Cell Cycle Progression.

Cell division entails a sequence of processes whose specific demands for biosynthetic precursors and energy place dynamic requirements on metabolism. However, little is known about how metabolic fluxes are coordinated with the cell division cycle. Here, we examine budding yeast to show that more than half of all measured metabolites change significantly through the cell division cycle. Cell cycle-dependent changes in central carbon metabolism are controlled by the cyclin-dependent kinase (Cdk1), a major cell cycle regulator, and the metabolic regulator protein kinase A. At the G1/S transition, Cdk1 phosphorylates and activates the enzyme Nth1, which funnels the storage carbohydrate trehalose into central carbon metabolism. Trehalose utilization fuels anabolic processes required to reliably complete cell division. Thus, the cell cycle entrains carbon metabolism to fuel biosynthesis. Because the oscillation of Cdk activity is a conserved feature of the eukaryotic cell cycle, we anticipate its frequent use in dynamically regulating metabolism for efficient proliferation.

Cyclin-Dependent Kinase Co-Ordinates Carbohydrate Metabolism and Cell Cycle in S. cerevisiae.

Cyclin-dependent kinases (CDKs) control cell division in eukaryotes by phosphorylating proteins involved in division. But successful proliferation requires co-ordination between division and cellular growth in mass. Previous proteomic studies suggested that metabolic proteins, as well as cell division proteins, could potentially be substrates of cyclin-dependent kinases. Here we focus on two metabolic enzymes of the yeast S. cerevisiae, neutral trehalase (Nth1) and glycogen phosphorylase (Gph1), and show that their activities are likely directly controlled by CDK activity, thus allowing co-ordinate regulation of carbohydrate metabolism with cell division processes. In this case, co-ordinate regulation may optimize the decision to undertake a final cell division as nutrients are being exhausted. Co-regulation of cell division processes and metabolic processes by CDK activity may be a general phenomenon important for co-ordinating the cell cycle with growth.

New Wnt/ß-catenin target genes promote experimental metastasis and migration of colorectal cancer cells through different signals.

OBJECTIVES: We have previously identified a 115-gene signature that characterises the metastatic potential of human primary colon cancers. The signature included the canonical Wnt target gene BAMBI, which promoted experimental metastasis in mice. Here, we identified three new direct Wnt target genes from the signature, and studied their functions in epithelial-mesenchymal transition (EMT), cell migration and experimental metastasis. DESIGN: We examined experimental liver metastases following injection of selected tumour cells into spleens of NOD/SCID mice. Molecular and cellular techniques were used to identify direct transcription target genes of Wnt/ß-catenin signals. Microarray analyses and experiments that interfered with cell migration through inhibitors were performed to characterise downstream signalling systems. RESULTS: Three new genes from the colorectal cancer (CRC) metastasis signature, BOP1, CKS2 and NFIL3, were identified as direct transcription targets of ß-catenin/TCF4. Overexpression and knocking down of these genes in CRC cells promoted and inhibited, respectively, experimental metastasis in mice, EMT and cell motility in culture. Cell migration was repressed by interfering with distinct signalling systems through inhibitors of PI3K, JNK, p38 mitogen-activated protein kinase and/or mTOR. Gene expression profiling identified a series of migration-promoting genes, which were induced by BOP1, CKS2 and NFIL3, and could be repressed by inhibitors that are specific to these pathways. CONCLUSIONS: We identified new direct Wnt/ß-catenin target genes, BOP1, CKS2 and NFIL3, which induced EMT, cell migration and experimental metastasis of CRC cells. These genes crosstalk with different downstream signalling systems, and activate migration-promoting genes. These pathways and downstream genes may serve as therapeutic targets in the treatment of CRC metastasis.

EVALUATION OF EFFECTIVENESS OF SYNCHRONIZATION METHODS OF CELL DIVISION IN YEAST SACCHAROMYCES CEREVISIAE.

Synchronization of cell division in cultures of yeast Saccharomyces cerevisiae is widely used in research on the regulation of gene expression and biochemical processes in eukaryotes at different stages of the cell cycle. Here, we compare the efficiency of modern most commonly used methods to achieve and assess the degree of synchronization of cell division in yeast. Block-and-release methods with alpha-factor, hydroxyurea, nocodazole, cdc28-4 mutation are described in detail with practical notes.

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 (Tyr(19)). 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.

Yeast as a model system to screen purine derivatives against human CDK1 and CDK2 kinases.

Cyclin-dependent kinases (Cdk) play crucial roles in cell cycle progression. Aberrant activation of Cdk1 has been observed in a number of primary tumors and Cdk2 is deregulated in various malignancies. The therapeutic value of targeting Cdk1 and Cdk2 has been explored in a number of experimental systems. In the present study, taking advantage of the fact that deletion of the yeast CDC28 gene is functionally complemented by human CDK1 or CDK2, we set up an in vivo screen system to evaluate the inhibitory potency of purine derivatives against these two human Cdks. We constructed three isogenic strains highly sensitive to small molecules and harboring genes CDK1, CDK2 or CDC28, under the control of the CDC28 promoter. In a proof of principle assay, we determined the inhibitory effect of 82 purine derivatives on the growth rate of these strains. Thirty-three of them were revealed to be able to inhibit the Cdk1- or Cdk2-harboring strains but not the Cdc28-harboring strain, suggesting a specific inhibitory effect on human Cdks. Our data demonstrate that the yeast-based assay is an efficient system to identify potential specific inhibitors that should be preferentially selected for further investigation in cultured human cell lines.

A Whi7-anchored loop controls the G1 Cdk-cyclin complex at start.

Cells commit to a new cell cycle at Start by activation of the G1 Cdk-cyclin complex which, in turn, triggers a genome-wide transcriptional wave that executes the G1/S transition. In budding yeast, the Cdc28-Cln3 complex is regulated by an ER-retention mechanism that is important for proper cell size control. We have isolated small-cell-size CDC28 mutants showing impaired retention at the ER and premature accumulation of the Cln3 cyclin in the nucleus. The differential interactome of a quintuple Cdc28(wee) mutant pinpointed Whi7, a Whi5 paralog targeted by Cdc28 that associates to the ER in a phosphorylation-dependent manner. Our results demonstrate that the Cln3 cyclin and Whi7 act in a positive feedback loop to release the G1 Cdk-cyclin complex and trigger Start once a critical size has been reached, thus uncovering a key nonlinear mechanism at the earliest known events of cell-cycle entry.