Cyclin-dependent Kinase Cdc28 Research Articles

Proteostatic tuning underpins the evolution of novel multicellular traits.

The evolution of multicellularity paved the way for the origin of complex life on Earth, but little is known about the mechanistic basis of early multicellular evolution. Here, we examine the molecular basis of multicellular adaptation in the multicellularity long-term evolution experiment (MuLTEE). We demonstrate that cellular elongation, a key adaptation underpinning increased biophysical toughness and organismal size, is convergently driven by down-regulation of the chaperone Hsp90. Mechanistically, Hsp90-mediated morphogenesis operates by destabilizing the cyclin-dependent kinase Cdc28, resulting in delayed mitosis and prolonged polarized growth. Reinstatement of Hsp90 or Cdc28 expression resulted in shortened cells that formed smaller groups with reduced multicellular fitness. Together, our results show how ancient protein folding systems can be tuned to drive rapid evolution at a new level of biological individuality by revealing novel developmental phenotypes.

Glucose to lactate shift reprograms CDK-dependent mitotic decisions and its communication with MAPK Sty1 in Schizosaccharomyces pombe.

Cell cycle regulation in response to biochemical cues is a fundamental event associated with many diseases. The regulation of such responses in complex metabolic environments is poorly understood. This study reveals unknown aspects of the metabolic regulation of cell division in Schizosaccharomyces pombe. We show that changing the carbon source from glucose to lactic acid alters the functions of the cyclin-dependent kinase (CDK) Cdc2 and mitogen-activated protein kinase (MAPK) Sty1, leading to unanticipated outcomes in the behavior and fate of such cells. Functional communication of Cdc2 with Sty1 is known to be an integral part of the cellular response to aberrant Cdc2 activity in S. pombe. Our results show that cross-talk between Cdc2 and Sty1, and the consequent Sty1-dependent regulation of Cdc2 activity, appears to be compromised and the relationship between Cdc2 activity and mitotic timing is also reversed in the presence of lactate. We also show that the biochemical status of cells under these conditions is an important determinant of the altered molecular functions mentioned above as well as the altered behavior of these cells.

Strain variation in gene expression impact of hyphal cyclin Hgc1 in Candida albicans.

Formation of hyphae is a key virulence trait of the fungal pathogen Candida albicans. Hypha morphogenesis depends upon the cyclin Hgc1, which acts together with cyclin-dependent protein kinase Cdc28 to phosphorylate effectors that drive polarized growth. Hgc1 has also been implicated in gene regulation through its effects on 2 transcription factors, Efg1 and Ume6. Here, we report RNA-sequencing (RNA-seq) analysis of 2 pairs of hgc1Δ/Δ mutants and their respective wild-type strains, which lie in 2 different genetic backgrounds. We find that hgc1Δ/Δ mutations alter expression of 271 genes in both genetic backgrounds and 266 of those genes respond consistently with regard to up- or down-regulation. Consistency is similar to what has been observed with efg1Δ/Δ mutations and greater than observed with nrg1Δ/Δ mutations in these 2 backgrounds. The gene expression response includes genes under Efg1 control, as expected from prior studies. Hgc1-responsive genes also include ergosterol biosynthetic genes and bud neck-related genes, which may reflect interactions between Hgc1 and additional transcription factors as well as effects of Hgc1 on cellular length-to-width ratios.

Absence of Wee1 alters global transcriptional response to oxidative stress in Schizosaccharomyces pombe.

Stress response and checkpoint activation are the main determinants of cellular survival in adverse conditions. In Schizosaccharomyces pombe, these are controlled by the Mitogen Activated Protein Kinase Spc1 and the Cyclin dependent Kinase Cdc2 respectively. Cdc2 is regulated positively by Cdc25 and negatively by Wee1. Changes in Cdc2 activity can be sensed by Spc1 resulting in the modulation of mitotic timing by Spc1. Functional cross talks between cell cycle regulation and MAPK machinery during regulation of mitotic timing are well characterised but the presence of similar communication during stress response remains unexplored. In this study we report how the checkpoint activator kinase Wee1 can also influence the transcriptional response to oxidative stress. We show that deletion of Wee1 results in changes in gene expression of the cells, especially with respect to genes whose expression is known to be regulated by Spc1. These differences are seen in unperturbed cells as well as during oxidative stress. Moreover, such variations extend beyond what could be expected to occur due to the known enhanced Spc1 activity of these cells. This is the first depiction of the influence of Wee1 and consequently Cdc2 activity on transcriptional response to oxidative stress.

Bacillus subtilis KLBMPGC81 suppresses appressorium-mediated plant infection by altering the cell wall integrity signaling pathway and multiple cell biological processes in Magnaporthe oryzae.

Magnaporthe oryzae is one of the most destructive crop pathogens in the world, causing huge losses in rice harvest every year. Bacillus subtilis is a potential biocontrol agent that has been explored in many crop systems because it is a potent producer of bioactive compounds. However, the mechanisms by which these agents control rice blasts are not fully understood. We show that B. subtilis KLBMPGC81 (KC81) and its supernatant (SUP) have high antimicrobial activity against M. oryzae strain Guy11. To better exploit KC81 as a biocontrol agent, the mechanism by which KC81 suppresses rice blast pathogens was investigated. This study shows that KC81 SUP is effective in controlling rice blast disease. The SUP has a significant effect on suppressing the growth of M. oryzae and appressorium-mediated plant infection. KC81 SUP compromises cell wall integrity, microtubules and actin cytoskeleton, mitosis, and autophagy, all of which are required for M. oryzae growth, appressorium development, and host infection. We further show that the SUP reduces the activity of the cyclin-dependent kinase Cdc2 by enhancing the phosphorylation of Cdc2 Tyr 15, thereby impairing mitosis in M. oryzae cells. SUP induces the cell wall sensor MoWsc1 to activate the cell wall integrity pathway and Mps1 and Pmk1 mitogen-activated protein kinases. Taken together, our findings reveal that KC81 is an effective fungicide that suppresses M. oryzae growth, appressorium formation, and host infection by abnormally activating the cell wall integrity pathway, disrupting the cytoskeleton, mitosis, and autophagy.

A quantitative and spatial analysis of cell cycle regulators during the fission yeast cycle

We have carried out a systems-level analysis of the spatial and temporal dynamics of cell cycle regulators in the fission yeast Schizosaccharomyces pombe. In a comprehensive single-cell analysis, we have precisely quantified the levels of 38 proteins previously identified as regulators of the G2 to mitosis transition and of 7 proteins acting at the G1- to S-phase transition. Only 2 of the 38 mitotic regulators exhibit changes in concentration at the whole-cell level: the mitotic B-type cyclin Cdc13, which accumulates continually throughout the cell cycle, and the regulatory phosphatase Cdc25, which exhibits a complex cell cycle pattern. Both proteins show similar patterns of change within the nucleus as in the whole cell but at higher concentrations. In addition, the concentrations of the major fission yeast cyclin-dependent kinase (CDK) Cdc2, the CDK regulator Suc1, and the inhibitory kinase Wee1 also increase in the nucleus, peaking at mitotic onset, but are constant in the whole cell. The significant increase in concentration with size for Cdc13 supports the view that mitotic B-type cyclin accumulation could act as a cell size sensor. We propose a two-step process for the control of mitosis. First, Cdc13 accumulates in a size-dependent manner, which drives increasing CDK activity. Second, from mid-G2, the increasing nuclear accumulation of Cdc25 and the counteracting Wee1 introduce a bistability switch that results in a rapid rise of CDK activity at the end of G2 and thus, brings about an orderly progression into mitosis.

Transcription factor Atf1-dependent degradation of the mitotic cyclin Cdc13 is regulated by multiple factors in Schizosaccharomyces pombe.

The bZIP transcription factor Atf1 is a key player in the transcriptional programme of Schizosaccharomyces pombe cell cycle. It also controls both expression and degradation of mitotic cyclin Cdc13. Temporal regulation of these opposing functions of Atf1 is critical for fidelity of cell division. Our investigations revealed that an increase in the activity of mitogen-activated protein kinase (MAPK) Spc1 during mitotic exit and the consequent phosphorylation of Atf1 along with the prevailing high activity of cyclin-dependent kinase Cdc2 regulate Cdc13 degradation. Our results also indicate the possibility of a complex interplay between Cdc2 inhibitory kinase Wee1, the anaphase-promoting complex and Atf1 during mitotic exit. These observations provide evidence of new regulatory mechanisms of mitotic exit.

Regulation of CLB6 expression by the cytoplasmic deadenylase Ccr4 through its coding and 3’ UTR regions

RNA stability control contributes to the proper expression of gene products. Messenger RNAs (mRNAs) in eukaryotic cells possess a 5’ cap structure and the 3’ poly(A) tail which are important for mRNA stability and efficient translation. The Ccr4-Not complex is a major cytoplasmic deadenylase and functions in mRNA degradation. The CLB1-6 genes in Saccharomyces cerevisiae encode B-type cyclins which are involved in the cell cycle progression together with the cyclin-dependent kinase Cdc28. The CLB genes consist of CLB1/2, CLB3/4, and CLB5/6 whose gene products accumulate at the G2-M, S-G2, and late G1 phase, respectively. These Clb protein levels are thought to be mainly regulated by the transcriptional control and the protein stability control. Here we investigated regulation of CLB1-6 expression by Ccr4. Our results show that all CLB1-6 mRNA levels were significantly increased in the ccr4Δ mutant compared to those in wild-type cells. Clb1, Clb4, and Clb6 protein levels were slightly increased in the ccr4Δ mutant, but the Clb2, Clb3, and Clb5 protein levels were similar to those in wild-type cells. Since both CLB6 mRNA and Clb6 protein levels were most significantly increased in the ccr4Δ mutant, we further analyzed the cis-elements for the Ccr4-mediated regulation within CLB6 mRNA. We found that there were destabilizing sequences in both coding sequence and 3’ untranslated region (3’ UTR). The destabilizing sequences in the coding region were found to be both within and outside the sequences corresponding the cyclin domain. The CLB6 3’ UTR was sufficient for mRNA destabilization and decrease of the reporter GFP gene and this destabilization involved Ccr4. Our results suggest that CLB6 expression is regulated by Ccr4 through the coding sequence and 3’ UTR of CLB6 mRNA.

Expression dynamics of G1 cyclins in Saccharomyces cerevisiae revealed by two photon scanning number and brightness microscopy

Cell size homeostasis, which results from the coordination between cell growth and division, is a critical aspect of cell physiology and function in all species. The synchronization between these processes takes place in part in late G1 phase, termed Start in yeast, and marks irreversible commitment to division. Start timing depends heavily on the G1 cyclin, Cln3, which activates the Cdc28 cyclin dependent kinase. It is thought that Cln3-Cdc28 initiates Start by phosphorylation of the transcriptional repressor Whi5, causing its dissociation from the transcriptional activator complex, SBF, and export from the nucleus.

Integrative multi-omics profiling reveals cAMP-independent mechanisms regulating hyphal morphogenesis in Candida albicans.

Microbial pathogens grow in a wide range of different morphologies that provide distinct advantages for virulence. In the fungal pathogen Candida albicans, adenylyl cyclase (Cyr1) is thought to be a master regulator of the switch to invasive hyphal morphogenesis and biofilm formation. However, faster growing cyr1Δ/Δ pseudorevertant (PR) mutants were identified that form hyphae in the absence of cAMP. Isolation of additional PR mutants revealed that their improved growth was due to loss of one copy of BCY1, the negative regulatory subunit of protein kinase A (PKA) from the left arm of chromosome 2. Furthermore, hyphal morphogenesis was improved in some of PR mutants by multigenic haploinsufficiency resulting from loss of large regions of the left arm of chromosome 2, including global transcriptional regulators. Interestingly, hyphal-associated genes were also induced in a manner that was independent of cAMP. This indicates that basal protein kinase A activity is an important prerequisite to induce hyphae, but activation of adenylyl cyclase is not needed. Instead, phosphoproteomic analysis indicated that the Cdc28 cyclin-dependent kinase and the casein kinase 1 family member Yck2 play key roles in promoting polarized growth. In addition, integrating transcriptomic and proteomic data reveals hyphal stimuli induce increased production of key transcription factors that contribute to polarized morphogenesis.

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.

First person – Agamani Ghosal

ABSTRACTFirst Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping early-career researchers promote themselves alongside their papers. Agamani Ghosal is first author on ‘Communication between Cyclin dependent kinase Cdc2 and the Wis1-Spc1 MAPK pathway determines mitotic timing in Schizosaccharomyces pombe’, published in BiO. Agamani is a PhD student in the Cell Division lab of Dr Geetanjali Sundaram.

Analysis of localization of cell-cycle regulators in Neurospora crassa

Most fungi are multinucleated organisms. In some fungi, they have asynchronous nuclei in the same cytoplasm. We analyzed a cell-cycle regulation mechanism using a model fungus Neurospora crassa, which can make heterokaryon cells. G1/S cyclin CLN-1 and cyclin-dependent kinase CDC-2 were tagged with different fluorescence in different strains and expressed. By forming a heterokaryon strain of these, two different fluorescence-tagged proteins were expressed in the same cytoplasm. CDC-2 was localized in all nuclei, whereas CLN-1 was not detected in most of the nuclei and was dispersed in the cytoplasm with small granular clusters. This indicates that in multinucleated fungi, cell-cycle regulators, similar to other proteins, are shared around the nuclei regardless of different cell-cycle stages. Moreover, each nucleus can select and use a special cell-cycle regulator only when it is necessary. Fungal nuclei may have a novel pickup mechanism of necessary proteins from their cytoplasm at the point of use.

Communication between Cyclin-dependent kinase Cdc2 and the Wis1-Spc1 MAPK pathway determines mitotic timing in Schizosaccharomyces pombe.

ABSTRACTCheckpoint activation and gene expression modulation represent key determinants of cellular survival in adverse conditions. The former is regulated by cyclin-dependent kinases (CDKs) while the latter can be controlled by mitogen-activated protein kinases (MAPKs). Association between cell-cycle progression and MAPK-dependent gene expression exists in cells growing in optimal environments. While MAPK-mediated regulation of the cell cycle is well characterised, the reciprocal influence of mitotic CDK on stress response is not well studied. We present evidence that CDK activity can regulate the extent of MAPK activation in Schizosaccharomyces pombe cells. We show that increasing or decreasing mitotic CDK (Cdc2) activity in S. pombe cells can affect the activation of stress responsive MAPK (Spc1) even in the absence of stress stimuli. Our results indicate that the strong correlation between Cdc2 activity and Spc1 MAPK-activity in S. pombe is important in regulating mitotic timing.This article has an associated First Person interview with the first author of the paper.

Discovery and optimization of novel pyridines as highly potent and selective glycogen synthase kinase 3 inhibitors

Glycogen synthase kinase-3 plays an essential role in multiple biochemical pathways in the cell, particularly in regards to energy regulation. As such, Glycogen synthase kinase-3 is an attractive target for pharmacological intervention in a variety of disease states, particularly non-insulin dependent diabetes mellitus. However, due to homology with other crucial kinases, such as the cyclin-dependent protein kinase CDC2, developing compounds that are both potent and selective is challenging. A novel series of derivatives of 5-nitro-N2-(2-(pyridine-2ylamino)ethyl)pyridine-2,6-diamine were synthesized and have been shown to potently inhibit glycogen synthase kinase-3 (GSK3). Potency in the low nanomolar range was obtained along with remarkable selectivity. The compounds activate glycogen synthase in insulin receptor-expressing CHO-IR cells and in primary rat hepatocytes, and have acceptable pharmacokinetics and pharmacodynamics to allow for oral dosing. The X-ray co-crystal structure of human GSK3-β in complex with compound 2 is reported and provides insights into the structural determinants of the series responsible for its potency and selectivity.

CDK phosphorylates the polarisome scaffold Spa2 to maintain its localization at the site of cell growth.

Polarisome is a protein complex that plays an important role in polarized growth in fungi by assembling actin cables towards the site of cell growth. For proper morphogenesis, the polarisome must localize to the right place at the right time. However, the mechanisms that control polarisome localization remain poorly understood. In this study, using the polymorphic fungus Candida albicans as a model, we have discovered that the cyclin-dependent kinase (CDK) Cdc28 phosphorylates the polarisome scaffold protein Spa2 to govern polarisome localization during both yeast and hyphal growth. In a yeast cell cycle, Cdc28-Clb2 phosphorylates Spa2 and controls the timing of polarisome translocation from the bud tip to the bud neck. And during hyphal development, Cdc28-Clb2 and the hyphal-specific Cdc28-Hgc1 cooperate to enhance Spa2 phosphorylation to maintain the polarisome at the hyphal tip. Blocking the CDK phosphorylation causes premature tip-to-neck translocation of Spa2 during yeast growth and inappropriate septal localization of Spa2 in hyphae and abnormal hyphal morphology under certain inducing conditions. Together, our results generate new insights into the mechanisms by which fungi regulate polarisome localization in the control of polarized growth.

Hgc1-Cdc28-how much does a single protein kinase do in the regulation of hyphal development in Candida albicans?

The fungal human pathogen Candida albicans can cause invasive infection with high mortality rates. A key virulence factor is its ability to switch between three morphologies: yeast, pseudohyphae and hyphae. In contrast to the ovalshaped unicellular yeast cells, hyphae are highly elongated, tube-like, and multicellular. A long-standing question is what coordinates all the cellular machines to construct cells with distinct shapes. Hyphal-specific genes (HSGs) are thought to hold the answer. Among the numerous HSGs found, only UME6 and HGC1 are required for hyphal development. UME6 encodes a transcription factor that regulates many HSGs including HGC1. HGC1 encodes a G1 cyclin which partners with the Cdc28 cyclin-dependent kinase. Hgc1-Cdc28 simultaneously phosphorylates and regulates multiple substrates, thus controlling multiple cellular apparatuses for morphogenesis. This review is focused on major progresses made in the past decade on Hgc1's roles and regulation in C. albicans hyphal development and other traits important for infection.

The Gcn2 Regulator Yih1 Interacts with the Cyclin Dependent Kinase Cdc28 and Promotes Cell Cycle Progression through G2/M in Budding Yeast.

The Saccharomyces cerevisiae protein Yih1, when overexpressed, inhibits the eIF2 alpha kinase Gcn2 by competing for Gcn1 binding. However, deletion of YIH1 has no detectable effect on Gcn2 activity, suggesting that Yih1 is not a general inhibitor of Gcn2, and has no phenotypic defect identified so far. Thus, its physiological role is largely unknown. Here, we show that Yih1 is involved in the cell cycle. Yeast lacking Yih1 displays morphological patterns and DNA content indicative of a delay in the G2/M phases of the cell cycle, and this phenotype is independent of Gcn1 and Gcn2. Accordingly, the levels of phosphorylated eIF2α, which show a cell cycle-dependent fluctuation, are not altered in cells devoid of Yih1. We present several lines of evidence indicating that Yih1 is in a complex with Cdc28. Yih1 pulls down endogenous Cdc28 in vivo and this interaction is enhanced when Cdc28 is active, suggesting that Yih1 modulates the function of Cdc28 in specific stages of the cell cycle. We also demonstrate, by Bimolecular Fluorescence Complementation, that endogenous Yih1 and Cdc28 interact with each other, confirming Yih1 as a bona fide Cdc28 binding partner. Amino acid substitutions within helix H2 of the RWD domain of Yih1 enhance Yih1-Cdc28 association. Overexpression of this mutant, but not of wild type Yih1, leads to a phenotype similar to that of YIH1 deletion, supporting the view that Yih1 is involved through Cdc28 in the regulation of the cell cycle. We further show that IMPACT, the mammalian homologue of Yih1, interacts with CDK1, the mammalian counterpart of Cdc28, indicating that the involvement with the cell cycle is conserved. Together, these data provide insights into the cellular function of Yih1/IMPACT, and provide the basis for future studies on the role of this protein in the cell cycle.

Morphogenesis checkpoint kinase Swe1 is the executor of lipolysis-dependent cell-cycle progression

Cell growth and division requires the precise duplication of cellular DNA content but also of membranes and organelles. Knowledge about the cell-cycle-dependent regulation of membrane and storage lipid homeostasis is only rudimentary. Previous work from our laboratory has shown that the breakdown of triacylglycerols (TGs) is regulated in a cell-cycle-dependent manner, by activation of the Tgl4 lipase by the major cyclin-dependent kinase Cdc28. The lipases Tgl3 and Tgl4 are required for efficient cell-cycle progression during the G1/S (Gap1/replication phase) transition, at the onset of bud formation, and their absence leads to a cell-cycle delay. We now show that defective lipolysis activates the Swe1 morphogenesis checkpoint kinase that halts cell-cycle progression by phosphorylation of Cdc28 at tyrosine residue 19. Saturated long-chain fatty acids and phytosphingosine supplementation rescue the cell-cycle delay in the Tgl3/Tgl4 lipase-deficient strain, suggesting that Swe1 activity responds to imbalanced sphingolipid metabolism, in the absence of TG degradation. We propose a model by which TG-derived sphingolipids are required to activate the protein phosphatase 2A (PP2A(Cdc55)) to attenuate Swe1 phosphorylation and its inhibitory effect on Cdc28 at the G1/S transition of the cell cycle.

Cell cycle-independent phospho-regulation of Fkh2 during hyphal growth regulates Candida albicans pathogenesis.

The opportunistic human fungal pathogen, Candida albicans, undergoes morphological and transcriptional adaptation in the switch from commensalism to pathogenicity. Although previous gene-knockout studies have identified many factors involved in this transformation, it remains unclear how these factors are regulated to coordinate the switch. Investigating morphogenetic control by post-translational phosphorylation has generated important regulatory insights into this process, especially focusing on coordinated control by the cyclin-dependent kinase Cdc28. Here we have identified the Fkh2 transcription factor as a regulatory target of both Cdc28 and the cell wall biosynthesis kinase Cbk1, in a role distinct from its conserved function in cell cycle progression. In stationary phase yeast cells 2D gel electrophoresis shows that there is a diverse pool of Fkh2 phospho-isoforms. For a short window on hyphal induction, far before START in the cell cycle, the phosphorylation profile is transformed before reverting to the yeast profile. This transformation does not occur when stationary phase cells are reinoculated into fresh medium supporting yeast growth. Mass spectrometry and mutational analyses identified residues phosphorylated by Cdc28 and Cbk1. Substitution of these residues with non-phosphorylatable alanine altered the yeast phosphorylation profile and abrogated the characteristic transformation to the hyphal profile. Transcript profiling of the phosphorylation site mutant revealed that the hyphal phosphorylation profile is required for the expression of genes involved in pathogenesis, host interaction and biofilm formation. We confirmed that these changes in gene expression resulted in corresponding defects in pathogenic processes. Furthermore, we identified that Fkh2 interacts with the chromatin modifier Pob3 in a phosphorylation-dependent manner, thereby providing a possible mechanism by which the phosphorylation of Fkh2 regulates its specificity. Thus, we have discovered a novel cell cycle-independent phospho-regulatory event that subverts a key component of the cell cycle machinery to a role in the switch from commensalism to pathogenicity.