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.

A precisely adjustable, variation-suppressed eukaryotic transcriptional controller to enable genetic discovery.

Conditional expression of genes and observation of phenotype remain central to biological discovery. Current methods enable either on/off or imprecisely controlled graded gene expression. We developed a 'well-tempered' controller, WTC846, for precisely adjustable, graded, growth condition independent expression of genes in Saccharomyces cerevisiae. Controlled genes are expressed from a strong semisynthetic promoter repressed by the prokaryotic TetR, which also represses its own synthesis; with basal expression abolished by a second, 'zeroing' repressor. The autorepression loop lowers cell-to-cell variation while enabling precise adjustment of protein expression by a chemical inducer. WTC846 allelic strains in which the controller replaced the native promoters recapitulated known null phenotypes (CDC42, TPI1), exhibited novel overexpression phenotypes (IPL1), showed protein dosage-dependent growth rates and morphological phenotypes (CDC28, TOR2, PMA1 and the hitherto uncharacterized PBR1), and enabled cell cycle synchronization (CDC20). WTC846 defines an 'expression clamp' allowing protein dosage to be adjusted by the experimenter across the range of cellular protein abundances, with limited variation around the setpoint.

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.

Stress granules display bistable dynamics modulated by Cdk.

Stress granules (SGs) are conserved biomolecular condensates that originate in response to many stress conditions. These membraneless organelles contain nontranslating mRNAs and a diverse subproteome, but our knowledge of their regulation and functional relevance is still incipient. Here, we describe a mutual-inhibition interplay between SGs and Cdc28, the budding yeast Cdk. Among Cdc28 interactors acting as negative modulators of Start, we have identified Whi8, an RNA-binding protein that localizes to SGs and recruits the mRNA of CLN3, the most upstream G1 cyclin, for efficient translation inhibition and Cdk inactivation under stress. However, Whi8 also contributes to recruiting Cdc28 to SGs, where it acts to promote their dissolution. As predicted by a mutual-inhibition framework, the SG constitutes a bistable system that is modulated by Cdk. Since mammalian cells display a homologous mechanism, we propose that the opposing functions of specific mRNA-binding proteins and Cdk's subjugate SG dynamics to a conserved hysteretic switch.

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 Phosphorylation of the Dam1 Complex Strengthens Kinetochore-Microtubule Attachments.

To ensure the faithful inheritance of DNA, a macromolecular protein complex called the kinetochore sustains the connection between chromosomes and force-generating dynamic microtubules during cell division. Defects in this process lead to aneuploidy, a common feature of cancer cells and the cause of many developmental diseases [1-4]. One of the major microtubule-binding activities in the kinetochore is mediated by the conserved Ndc80 complex (Ndc80c) [5-7]. In budding yeast, the retention of kinetochores on dynamic microtubule tips also depends on the essential heterodecameric Dam1 complex (Dam1c) [8-15], which binds to the Ndc80c and is proposed to be a functional ortholog of the metazoan Ska complex [16, 17]. The load-bearing activity of the Dam1c depends on its ability to oligomerize, and the purified complex spontaneously self-assembles into microtubule-encircling oligomeric rings, which are proposed to function as collars that allow kinetochores to processively track the plus-end tips of microtubules and harness the forces generated by disassembling microtubules [10-15, 18-22]. However, it is unknown whether there are specific regulatory events that promote Dam1c oligomerization to ensure accurate segregation. Here, we used a reconstitution system to discover that Cdk1, the major mitotic kinase that drives the cell cycle, phosphorylates the Ask1 component of the Dam1c to increase its residence time on microtubules and enhance kinetochore-microtubule attachment strength. We propose that Cdk1 activity promotes Dam1c oligomerization to ensure that kinetochore-microtubule attachments are stabilized as kinetochores come under tension in mitosis.

The Multiple Roles of the Cdc14 Phosphatase in Cell Cycle Control.

The Cdc14 phosphatase is a key regulator of mitosis in the budding yeast Saccharomyces cerevisiae. Cdc14 was initially described as playing an essential role in the control of cell cycle progression by promoting mitotic exit on the basis of its capacity to counteract the activity of the cyclin-dependent kinase Cdc28/Cdk1. A compiling body of evidence, however, has later demonstrated that this phosphatase plays other multiple roles in the regulation of mitosis at different cell cycle stages. Here, we summarize our current knowledge about the pivotal role of Cdc14 in cell cycle control, with a special focus in the most recently uncovered functions of the phosphatase.

Competition in the chaperone-client network subordinates cell-cycle entry to growth and stress.

The precise coordination of growth and proliferation has a universal prevalence in cell homeostasis. As a prominent property, cell size is modulated by the coordination between these processes in bacterial, yeast, and mammalian cells, but the underlying molecular mechanisms are largely unknown. Here, we show that multifunctional chaperone systems play a concerted and limiting role in cell-cycle entry, specifically driving nuclear accumulation of the G1 Cdk-cyclin complex. Based on these findings, we establish and test a molecular competition model that recapitulates cell-cycle-entry dependence on growth rate. As key predictions at a single-cell level, we show that availability of the Ydj1 chaperone and nuclear accumulation of the G1 cyclin Cln3 are inversely dependent on growth rate and readily respond to changes in protein synthesis and stress conditions that alter protein folding requirements. Thus, chaperone workload would subordinate Start to the biosynthetic machinery and dynamically adjust proliferation to the growth potential of the cell.

Cdc14 activation requires coordinated Cdk1-dependent phosphorylation of Net1 and PP2A-Cdc55 at anaphase onset.

Exit from mitosis and completion of cytokinesis require the inactivation of mitotic cyclin-dependent kinase (Cdk) activity. In budding yeast, Cdc14 phosphatase is a key mitotic regulator that is activated in anaphase to counteract Cdk activity. In metaphase, Cdc14 is kept inactive in the nucleolus, where it is sequestered by its inhibitor, Net1. At anaphase onset, downregulation of PP2ACdc55 phosphatase by separase and Zds1 protein promotes Net1 phosphorylation and, consequently, Cdc14 release from the nucleolus. The mechanism by which PP2ACdc55 activity is downregulated during anaphase remains to be elucidated. Here, we demonstrate that Cdc55 regulatory subunit is phosphorylated in anaphase in a Cdk1-Clb2-dependent manner. Interestingly, cdc55-ED phosphomimetic mutant inactivates PP2ACdc55 phosphatase activity towards Net1 and promotes Cdc14 activation. Separase and Zds1 facilitate Cdk-dependent Net1 phosphorylation and Cdc14 release from the nucleolus by modulating PP2ACdc55 activity via Cdc55 phosphorylation. In addition, human Cdk1-CyclinB1 phosphorylates human B55, indicating that the mechanism is conserved in higher eukaryotes.

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.

Cyclin-dependent kinase modulates budding yeast Rad5 stability during cell cycle.

The DNA damage tolerance (DDT) pathway facilitates the bypass of the fork-blocking lesions without removing them through either translesion DNA synthesis or error-free damage bypass mechanism. The Saccharomyces cerevisiae Rad5 is a multi-functional protein involved in the error-free branch of the DDT pathway, and its protein level periodically fluctuates through the cell cycle; however, the mechanistic basis and functional importance of the Rad5 level for the cell cycle regulation remain unclear. Here, we show that Rad5 is predominantly phosphorylated on serine 130 (S130) during S/G2 phase and that this modification depends on the cyclin-dependent kinase Cdc28/CDK1. We also show that the phosphorylated Rad5 species at S130 exhibit a relatively short half-life compared with non-phosphorylated Rad5 moiety, and that the Rad5 protein is partially stabilized in phosphorylation-defective rad5 S130A cells. Importantly, the elimination of this modification results in a defective cell-cycle dependent Rad5 oscillation pattern. Together, our results demonstrate that CDK1 modulates Rad5 stability by phosphorylation during the cell cycle, suggesting a crosstalk between the phosphorylation and degradation of Rad5.

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.

High levels of histones promote whole-genome-duplications and trigger a Swe1WEE1-dependent phosphorylation of Cdc28CDK1.

Whole-genome duplications (WGDs) have played a central role in the evolution of genomes and constitute an important source of genome instability in cancer. Here, we show in Saccharomyces cerevisiae that abnormal accumulations of histones are sufficient to induce WGDs. Our results link these WGDs to a reduced incorporation of the histone variant H2A.Z to chromatin. Moreover, we show that high levels of histones promote Swe1WEE1 stabilisation thereby triggering the phosphorylation and inhibition of Cdc28CDK1 through a mechanism different of the canonical DNA damage response. Our results link high levels of histones to a specific type of genome instability that is quite frequently observed in cancer and uncovers a new mechanism that might be able to respond to high levels of histones.

Clb6-Cdc28 Promotes Ribonucleotide Reductase Subcellular Redistribution during S Phase.

A tightly controlled cellular deoxyribonucleotide (deoxynucleoside triphosphate [dNTP]) pool is critical for maintenance of genome integrity. One mode of dNTP pool regulation is through subcellular localization of ribonucleotide reductase (RNR), the enzyme that catalyzes the rate-limiting step of dNTP biosynthesis. In Saccharomyces cerevisiae, the RNR small subunit, Rnr2-Rnr4, is localized to the nucleus, whereas the large subunit, Rnr1, is cytoplasmic. As cells enter S phase or encounter DNA damage, Rnr2-Rnr4 relocalizes to the cytoplasm to form an active holoenzyme complex with Rnr1. Although the DNA damage-induced relocalization requires the checkpoint kinases Mec1-Rad53-Dun1, the S-phase-specific redistribution does not. Here, we report that the S-phase cyclin-cyclin-dependent kinase (CDK) complex Clb6-Cdc28 controls Rnr2-Rnr4 relocalization in S phase. Rnr2 contains a consensus CDK site and exhibits Clb6-dependent phosphorylation in S phase. Deletion of CLB6 or removal of the CDK site results in an increased association of Rnr2 with its nuclear anchor Wtm1, nuclear retention of Rnr2-Rnr4, and an enhanced sensitivity to the RNR inhibitor hydroxyurea. Thus, we propose that Rnr2-Rnr4 redistribution in S phase is triggered by Clb6-Cdc28-mediated phosphorylation of Rnr2, which disrupts the Rnr2-Wtm1 interaction and promotes the release of Rnr2-Rnr4 from the nucleus.

The Cks1/Cks2 axis fine-tunes Mll1 expression and is crucial for MLL-rearranged leukaemia cell viability.

The Cdc28 protein kinase subunits, Cks1 and Cks2, play dual roles in Cdk-substrate specificity and Cdk-independent protein degradation, in concert with the E3 ubiquitin ligase complexes SCFSkp2 and APCCdc20. Notable targets controlled by Cks include p27 and Cyclin A. Here, we demonstrate that Cks1 and Cks2 proteins interact with both the MllN and MllC subunits of Mll1 (Mixed-lineage leukaemia 1), and together, the Cks proteins define Mll1 levels throughout the cell cycle. Overexpression of CKS1B and CKS2 is observed in multiple human cancers, including various MLL-rearranged (MLLr) AML subtypes. To explore the importance of MLL-Fusion Protein regulation by CKS1/2, we used small molecule inhibitors (MLN4924 and C1) to modulate their protein degradation functions. These inhibitors specifically reduced the proliferation of MLLr cell lines compared to primary controls. Altogether, this study uncovers a novel regulatory pathway for MLL1, which may open a new therapeutic approach to MLLr leukaemia.

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.