Behind the wheel and under the hood: Functions of cyclin-dependent kinases in response to DNA damage

Cell division and the response to genotoxic stress are intimately connected in eukaryotes, for example, by checkpoint pathways that signal the presence of DNA damage or its ongoing repair to the cell cycle machinery, leading to reversible arrest or apoptosis. Recent studies reveal another connection: the cyclin-dependent kinases (CDKs) that govern both DNA synthesis (S) phase and mitosis directly coordinate DNA repair processes with progression through the cell cycle. In both mammalian cells and yeast, the two major modes of double strand break (DSB) repair – homologous recombination (HR) and non-homologous end joining (NHEJ) – are reciprocally regulated during the cell cycle. In yeast, the cell cycle kinase Cdk1 directly promotes DSB repair by HR during the G2 phase. In mammalian cells, loss of Cdk2, which is active throughout S and G2 phases, results in defective DNA damage repair and checkpoint signaling. Here we provide an overview of data that implicate CDKs in the regulation of DNA damage responses in yeast and metazoans. In yeast, CDK activity is required at multiple points in the HR pathway; the precise roles of CDKs in mammalian HR have yet to be determined. Finally, we consider how the two different, and in some cases opposing, roles of CDKs – as targets of negative regulation by checkpoint signaling and as positive effectors of repair pathway selection and function – could be balanced to produce a coordinated and effective response to DNA damage.     

Novel plant-specific cyclin-dependent kinase inhibitors induced by biotic and abiotic stresses

The EL2 gene of rice (Oryza sativa), previously classified as early response gene against the potent biotic elicitor N-acetylchitoheptaose and encoding a short polypeptide with unknown function, was identified as a novel cell cycle regulatory gene related to the recently reported SIAMESE (SIM) gene of Arabidopsis thaliana. Iterative two-hybrid screens, in vitro pull-down assays, and fluorescence resonance energy transfer analyses showed that Orysa; EL2 binds the cyclin-dependent kinase (CDK) CDKA1;1 and D-type cyclins. No interaction was observed with the plant-specific B-type CDKs. The amino acid motif ELERFL was identified to be essential for cyclin, but not for CDK binding. Orysa;EL2 impaired the ability of Orysa; CYCD5;3 to complement a budding yeast (Saccharomyces cerevisiae) triple CLN mutant, whereas recombinant protein inhibited CDK activity in vitro. Moreover, Orysa;EL2 was able to rescue the multicellular trichome phenotype of sim mutants of Arabidopsis, unequivocally demonstrating that Orysa;EL2 operates as a cell cycle inhibitor. Orysa;EL2 mRNA levels were induced by cold, drought, and propionic acid. Our data suggest that Orysa;EL2 encodes a new type of plant CDK inhibitor that links cell cycle progression with biotic and abiotic stress responses.     

Silencing of CDK2, but not CDK1, separates mitogenic from anti-apoptotic signaling,sensitizing p53 defective cells for synthetic lethality

Small molecule inhibitors targeting CDK1/CDK2 have been clinically proven effective against a variety of tumors, albeit at the cost of profound off target toxicities. To separate potential therapeutic from toxic effects, we selectively knocked down CDK1 or CDK2 in p53 mutated HACAT cells by siRNA silencing. Using dynamic, cell cycle wide proteome arrays, we observed minor changes in overall abundance of proteins critically involved in cell cycle transition despite profound G₂/M or G₁/S arrest, respectively. Employing phospho site specific analyses, we identified uncoupled mitogenic, yet pro-apoptotic signaling from counter balancing anti-apoptotic activity in CDK2 disrupted cells. Moreover, a crucial role of CDK2 activity in early serum response was observed, extending well-established roles of CDKs outside their cell cycle regulating functions. In contrast, disruption of CDK1 only marginally affected phosphorylation events of crucial signaling nodes prior to G₂/S transition. The data presented here suggest that the temporal separation of pro- and anti-apoptotic pathways by selective inhibition of CDK2 disrupts coherent signaling modules and may synergize with anti-proliferative drugs, averting toxic side effects from CDK1 inhibition.     

Cyclin dependent kinase regulation

Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle and their activities are consequently tightly regulated. Recent developments in the field of CDK regulation have included the discovery and characterization of CDK inhibitors. These developments have had an impact on our understanding of how other signalling pathways may be linked to the cell cycle machinery.     

A plant solution to the CDK conundrum in the DNA damage response

To cope with DNA damage, proliferating cells have evolved sophisticated mechanisms including cell cycle arrest and activation of DNA repair. Paradoxically, various DNA damage response pathways are promoted by cyclin‐dependent kinase (CDK) activity, while cell cycle remains arrested. New work in The EMBO Journal shows that plant cells have evolved intricate ways to resolve this dilemma, by utilizing distinct and specialized CDKs for cell cycle progression and homologous recombination.     

Regulation of cyclin-dependent kinases in Arabidopsis thaliana

In plants, different families of cyclin-dependent kinases (CDKs) and cyclins have been identified, indicating that also in plants the progression through the cell cycle is regulated by CDKs. In all eukaryotes, CDKs exert their activity through well-controlled phosphorylations of specific substrates on serine/threonine residues. Such post-translational modifications are universal mechanisms in signal transduction pathways. They allow the organism to differentiate, regulate growth and/or adapt to environmental changes, the latter being crucial for plants because of their sedentary life-style. This adaptation might explain the occurrence of a special CDK type with plant-specific features. This review focuses on the involvement of plant CDKs in different phases of the cell cycle in Arabidopsis thaliana and outlines their regulation by binding to other proteins, and by phosphorylation and dephosphorylation.     

Plant cell cycle transitions

Three decades have passed since the first recognition of restriction checkpoints in the plant cell cycle. Although many core cell cycle genes have been cloned, the mechanisms that control the G1-->S and G2-->M transitions in plants have only recently started to be understood. The cyclin-dependent kinases (CDKs) play a central role in the regulation of the cell cycle, and the activity of these kinases is steered by regulatory subunits, the cyclins. The activities of CDK-cyclin complexes are further controlled by an intricate panoply of monitoring mechanisms, which result in oscillating CDK activity during the division cycle. These fluctuations trigger transitions between the different stages of the cell cycle.     

Analysis of differentially expressed genes in abiotic stress response and their role in signal transduction pathways

The study of abiotic stress response of plants is important because they have to cope with environmental changes to survive. The plant genomes have evolved to meet environmental challenges. Salt, temperature, and drought are the main abiotic stresses. The tolerance and response to stress vary differently in plants. The idea was to analyze the genes showing differential expression under abiotic stresses. There are many pathways connecting the perception of external stimuli to cellular responses. In plants, these pathways play an important role in the transduction of abiotic stresses. In the present study, the gene expression data have been analyzed for their involvement in different steps of signaling pathways. The conserved genes were analyzed for their role in each pathway. The functional annotations of these genes and their response under abiotic stresses in other plant species were also studied. The enzymes of signal pathways, showing similarity with conserved genes, were analyzed for their role in different abiotic stresses. Our findings will help to understand the expression of genes in response to various abiotic stresses. These genes may be used to study the response of different abiotic stresses in other plant species and the molecular basis of stress tolerance.     

Inactivation of the cyclin-dependent kinase Cdc28 abrogates cell cycle arrest induced by DNA damage and disassembly of mitotic spindles in Saccharomyces cerevisiae.

Eukaryotic cells may halt cell cycle progression following exposure to certain exogenous agents that damage cellular structures such as DNA or microtubules. This phenomenon has been attributed to functions of cellular control mechanisms termed checkpoints. Studies with the fission yeast Schizosaccharomyces pombe and mammalian cells have led to the conclusion that cell cycle arrest in response to inhibition of DNA replication or DNA damage is a result of down-regulation of the cyclin-dependent kinases (CDKs). Based on these studies, it has been proposed that inhibition of the CDK activity may constitute a general mechanism for checkpoint controls. Observations made with the budding yeast Saccharomyces cerevisiae, however, appear to disagree with this model. It has been shown that high levels of mitotic CDK activity are present in the budding yeast cells arrested in G2/mitosis as the result of DNA damage or replication inhibition. In this report, we show that a novel mutant allele of the CDC28 gene, encoding the budding yeast CDK, allowed cell cycle passage through mitosis and nuclear division in the presence of DNA damage and the microtubule toxin nocodazole at a restrictive temperature. Unlike the checkpoint-defective mutations in CDKs of fission yeast and mammalian cells, the cdc28 mutation that we identified was recessive and resulted in a loss of the CDK activity, including the Clb2-, Clb5-, and Clb6-associated, but not the Clb3-associated, CDK activities. Examination of several known alleles of cdc28 revealed that they were also, albeit partially, defective in cell cycle arrest in response to UV-generated DNA damage. These findings suggest that Cdc28 kinase in budding yeast may be required for cell cycle arrest resulting from DNA damage and disassembly of mitotic spindles.     

Exploring the selectivity of a ligand complex with CDK2/CDK1: a molecular dynamics simulation approach

Cyclin‐dependent kinases (CDKs) are core components of the cell cycle machinery that govern the transition between phases during cell cycle progression. Abnormalities in CDKs activity and regulation are common features of cancer, making CDK family members attractive targets for the development of anticancer drugs. Their inhibitors have entered in clinical trials to treat cancer. Very recently, Heathcote et al. (J. Med. Chem. 2010, 53:8508–8522) have found a ligand BS194 that has a high affinity with CDK2 (IC₅₀ = 3 nm ) but shows low affinity with CDK1 (IC₅₀ = 30 nm ). To understand the selectivity, we used homology modeling, molecular docking, molecular dynamics, and free‐energy calculation to analyze the interactions. A rational three‐dimensional model of the CDK1/BS194 complex is built. We found that Leu83 is a key residue that recognizes BS194 more effectively with CDK2 with good binding free energies rather than CDK1. Energetic analysis reveals that van der Waals interaction and non‐polar contributions to solvent are favorable in the formation of complexes and amine group of the ligand, which plays a crucial role for binding selectivity between CDK2 and CDK1. Copyright © 2012 John Wiley & Sons, Ltd.     

PP2A holoenzymes negatively and positively regulate cell cycle progression by dephosphorylating pocket proteins and multiple CDK substrates

Cell cycle progression is negatively regulated by the retinoblastoma family of pocket proteins and CDK inhibitors (CKIs). In contrast, CDKs promote progression through multiple phases of the cell cycle. One prominent way by which CDKs promote cell cycle progression is by inactivation of pocket proteins via hyperphosphorylation. Reactivation of pocket proteins to halt cell cycle progression requires dephosphorylation of multiple CDK-phosphorylated sites and is accomplished by PP2A and PP1 serine/threonine protein phosphatases. The same phosphatases are also implicated in dephosphorylation of multiple CDK substrates as cells exit mitosis and reenter the G1 phase of the cell cycle. This review is primarily focused on the role of PP2A and PP1 in the activation of pocket proteins during the cell cycle and in response to signaling cues that trigger cell cycle exit. Other functions of PP2A during the cell cycle will be discussed in brief, as comprehensive reviews on this topic have been published recently (De Wulf et al., 2009; Wurzenberger and Gerlich, 2011).     

The retinoblastoma pathway in plant cell cycle and development

The activity of cyclin-dependent kinases (CDKs) on specific targets mediates the temporal regulation of plant cell cycle transitions. The sequential activity of CDKs and the spatial regulation of cell proliferation during plant development, however, are still poorly understood. Understanding these aspects depends on the identification of the downstream targets and upstream modulators of CDKs and their regulation in response to mitogenic and/or differentiation signals. Current efforts to elucidate the answers to these questions are very promising; in particular, recent works reveal the essential role that the retinoblastoma pathway plays in controlling cell cycle progression and, presumably, some developmental events.     

Regulation of physiological aspects in plants by hydrogen sulfide and nitric oxide under challenging environment

Plants are exposed to a plethora of abiotic stresses such as drought, salinity, heavy metal and temperature stresses at different stages of their life cycle, from germination to seedling till the reproductive phase. As protective mechanisms, plants release signaling molecules that initiate a cascade of stress-signaling events, leading either to programmed cell death or plant acclimation. Hydrogen sulfide (H₂S) and nitric oxide (NO) are considered as new ‘gasotransmitter’ molecules that play key roles in regulating gene expression, posttranslational modification (PTM), as well as cross-talk with other hormones. Although the exact role of NO in plants remains unclear and is species dependent, various studies have suggested a positive correlation between NO accumulation and environmental stress in plants. These molecules are also involved in a large array of stress responses and act synergistically or antagonistically as signaling components, depending on their respective concentration. This study provides a comprehensive update on the signaling interplay between H₂S and NO in the regulation of various physiological processes under multiple abiotic stresses, modes of action and effects of exogenous application of these two molecules under drought, salt, heat and heavy metal stresses. However, the complete picture of the signaling cascades mediated by H₂S and NO is still elusive. Recent researches indicate that during certain plant processes, such as stomatal closure, H₂S could act upstream of NO signaling or downstream of NO in response to abiotic stresses by improving antioxidant activity in most plant species. In addition, PTMs of antioxidative pathways by these two molecules are also discussed.