Regulation of the cell cycle by protein phosphatase 2A in Saccharomyces cerevisiae.

Protein phosphatase 2A (PP2A) has long been implicated in cell cycle regulation in many different organisms. In the yeast Saccharomyces cerevisiae, PP2A controls cell cycle progression mainly through modulation of cyclin-dependent kinase (CDK) at the G(2)/M transition. However, CDK does not appear to be a direct target of PP2A. PP2A affects CDK activity through its roles in checkpoint controls. Inactivation of PP2A downregulates CDK by activating the morphogenesis checkpoint and, consequently, delays mitotic entry. Defects in PP2A also compromise the spindle checkpoint and predispose the cell to an error-prone mitotic exit. In addition, PP2A is involved in controlling the G(1)/S transition and cytokinesis. These findings suggest that PP2A functions in many stages of the cell cycle and its effect on cell cycle progression is pleiotropic.     

Cell cycle checkpoint regulators reach a zillion

Entry into mitosis is regulated by a checkpoint at the boundary between the G₂ and M phases of the cell cycle (G₂/M). In many organisms, this checkpoint surveys DNA damage and cell size and is controlled by both the activation of mitotic cyclin-dependent kinases (Cdks) and the inhibition of an opposing phosphatase, protein phosphatase 2A (PP2A). Misregulation of mitotic entry can often lead to oncogenesis or cell death. Recent research has focused on discovering the signaling pathways that feed into the core checkpoint control mechanisms dependent on Cdk and PP2A. Herein, we review the conserved mechanisms of the G₂/M transition, including recently discovered upstream signaling pathways that link cell growth and DNA replication to cell cycle progression. Critical consideration of the human, frog and yeast models of mitotic entry frame unresolved and emerging questions in this field, providing a prediction of signaling molecules and pathways yet to be discovered.     

Regulation of the female mouse germ cell cycle during entry into meiosis

During mouse embryonic development germ cells proliferate extensively until they commit to the male or female pathway and arrest in mitosis or meiosis respectively. Whilst the transition of female germ cells exiting the mitotic cell cycle and entering meiosis is well defined histologically, the essential cell cycle proteins involved in this process have remained unresolved. Using flow cytometry we have examined the entry of female germ cells into meiosis, their termination of DNA synthesis and entry into prophase I. Analysis of key G₂/M cell cycle proteins revealed that entry into meiosis and cell cycle exit at G₂/M involves repression of G₂/M promoting Cyclin B1, coincident upregulation of G₂/M repressing Cyclin B3 and robust establishment of the ATM/CHK2 pathway. By contrast we show that the ATR/CHK1 pathway is activated in male and female germ cells. This data indicates that an important G₂/M surveillance mechanism operates during germ cell proliferation and that passage into meiotic G₂/M involves the combined repression of G₂/M through Cyclin B3 and activation of the key G₂/M checkpoint regulatory network modulated through ATM and CHK2. This work shows that the core regulatory machinery that controls G₂/M progression in mitotic cells is activated in female mouse germ cells as they enter meiosis.     

Mitochondrial Fragmentation Due to Inhibition of Fusion Increases Cyclin B through Mitochondrial Superoxide Radicals

During the cell cycle, mitochondria undergo regulated changes in morphology. Two particularly interesting events are first, mitochondrial hyperfusion during the G₁-S transition and second, fragmentation during entry into mitosis. The mitochondria remain fragmented between late G₂- and mitotic exit. This mitotic mitochondrial fragmentation constitutes a checkpoint in some cell types, of which little is known. We bypass the ‘mitotic mitochondrial fragmentation’ checkpoint by inducing fragmented mitochondrial morphology and then measure the effect on cell cycle progression. Using Drosophila larval hemocytes, Drosophila S2R⁺ cell and cells in the pouch region of wing imaginal disc of Drosophila larvae we show that inhibiting mitochondrial fusion, thereby increasing fragmentation, causes cellular hyperproliferation and an increase in mitotic index. However, mitochondrial fragmentation due to over-expression of the mitochondrial fission machinery does not cause these changes. Our experiments suggest that the inhibition of mitochondrial fusion increases superoxide radical content and leads to the upregulation of cyclin B that culminates in the observed changes in the cell cycle. We provide evidence for the importance of mitochondrial superoxide in this process. Our results provide an insight into the need for mitofusin-degradation during mitosis and also help in understanding the mechanism by which mitofusins may function as tumor suppressors.     

Changes in expression of cell‐cycle‐related genes in PC‐3 prostate cancer cells caused by ovine uterine serpin

The hormonal‐regulated serpin, ovine uterine serpin (OvUS), also called uterine milk protein (UTMP), inhibits proliferation of lymphocytes and prostate cancer (PC‐3) cells by blocking cell‐cycle progression. The present aim was to identify cell‐cycle‐related genes regulated by OvUS in PC‐3 cells using the quantitative human cell‐cycle RT² Profiler? PCR array. Cells were cultured ±200 µg/ml recombinant OvUS (rOvUS) for 12 and 24 h. At 12 h, rOvUS increased expression of three genes related to cell‐cycle checkpoints and arrest (CDKN1A, CDKN2B, and CCNG2). Also, 14 genes were down‐regulated including genes involved in progression through S (MCM3, MCM5, PCNA), M (CDC2, CKS2, CCNH, BIRC5, MAD2L1, MAD2L2), G₁ (CDK4, CUL1, CDKN3) and DNA damage checkpoint and repair genes RAD1 and RBPP8. At 24 h, rOvUS decreased expression of 16 genes related to regulation and progression through M (BIRC5, CCNB1, CKS2, CDK5RAP1, CDC20, E2F4, MAD2L2) and G₁ (CDK4, CDKN3, TFDP2), DNA damage checkpoints and repair (RAD17, BRCA1, BCCIP, KPNA2, RAD1). Also, rOvUS down‐regulated the cell proliferation marker gene MKI67, which is absent in cells at G₀. Results showed that OvUS blocks cell‐cycle progression through upregulation of cell‐cycle checkpoint and arrest genes and down‐regulation of genes involved in cell‐cycle progression. J. Cell. Biochem. 107: 1182–1188, 2009. © 2009 Wiley‐Liss, Inc.     

Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) Protein Is Involved in Centrosome Separation through the Regulation of NIMA (Never In Mitosis Gene A)-related Kinase 2 (NEK2) Protein Activity

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is overexpressed in most human cancers and has been described as being involved in the progression of several human malignancies via the inhibition of protein phosphatase 2A (PP2A) activity toward c-Myc. However, with the exception of this role, the cellular function of CIP2A remains poorly understood. On the basis of yeast two-hybrid and coimmunoprecipitation assays, we demonstrate here that NIMA (never in mitosis gene A)-related kinase 2 (NEK2) is a binding partner for CIP2A. CIP2A exhibited dynamic changes in distribution, including the cytoplasm and centrosome, depending on the cell cycle stage. When CIP2A was depleted, centrosome separation and the mitotic spindle dynamics were impaired, resulting in the activation of spindle assembly checkpoint signaling and, ultimately, extension of the cell division time. Our data imply that CIP2A strongly interacts with NEK2 during G₂/M phase, thereby enhancing NEK2 kinase activity to facilitate centrosome separation in a PP1- and PP2A-independent manner. In conclusion, CIP2A is involved in cell cycle progression through centrosome separation and mitotic spindle dynamics.     

Dovitinib induces mitotic defects and activates the G2 DNA damage checkpoint

Dovitinib (TKI258; formerly CHIR‐258) is an orally bioavailable inhibitor of multiple receptor tyrosine kinases. Interestingly, Dovitinib triggered a G₂/M arrest in cancer cell lines from diverse origins including HeLa, nasopharyngeal carcinoma, and hepatocellular carcinoma. Single‐cell analysis revealed that Dovitinib promoted a delay in mitotic exit in a subset of cells, causing the cells to undergo mitotic slippage. Higher concentrations of Dovitinib induced a G₂ arrest similar to the G₂ DNA damage checkpoint. In support of this, DNA damage was triggered by Dovitinib as revealed by γ‐H2AX and comet assays. The mitotic kinase CDK1 was found to be inactivated by phosphorylation in the presence of Dovitinib. Furthermore, the G₂ arrest could be overcome by abrogation of the G₂ DNA damage checkpoint using small molecule inhibitors of CHK1 and WEE1. Finally, Dovitinib‐mediated G₂ cell cycle arrest and subsequent cell death could be promoted after DNA damage repair was disrupted by inhibitors of poly(ADP‐ribose) polymerases. These results are consistent with the recent finding that Dovitinib can also target topoisomerases. Collectively, these results suggest additional directions for use of Dovitinib, in particular with agents that target the DNA damage checkpoint.     

Protein phosphatase 2A (PP2A) promotes anaphase entry after DNA replication stress in budding yeast

DNA replication stress activates the S-phase checkpoint that arrests the cell cycle, but it is poorly understood how cells recover from this arrest. Cyclin-dependent kinase (CDK) and protein phosphatase 2A (PP2A) are key cell cycle regulators, and Cdc55 is a regulatory subunit of PP2A in budding yeast. We found that yeast cells lacking functional PP2A^Cdc55 showed slow growth in the presence of hydroxyurea (HU), a DNA synthesis inhibitor, without obvious viability loss. Moreover, PP2A mutants exhibited delayed anaphase entry and sustained levels of anaphase inhibitor Pds1 after HU treatment. A DNA damage checkpoint Chk1 phosphorylates and stabilizes Pds1. We show that chk1Δ and mutation of the Chk1 phosphorylation sites in Pds1 largely restored efficient anaphase entry in PP2A mutants after HU treatment. In addition, deletion of SWE1, which encodes the inhibitory kinase for CDK or mutation of the Swe1 phosphorylation site in CDK (cdc28F19), also suppressed the anaphase entry delay in PP2A mutants after HU treatment. Our genetic data suggest that Swe1/CDK acts upstream of Pds1. Surprisingly, cdc55Δ showed significant suppression to the viability loss of S-phase checkpoint mutants during DNA synthesis block. Together, our results uncover a PP2A-Swe1-CDK-Chk1-Pds1 axis that promotes recovery from DNA replication stress.     

EDD,a Ubiquitin-protein Ligase of the N-end Rule Pathway,Associates with Spindle Assembly Checkpoint Components and Regulates the Mitotic Response to Nocodazole

In this work, we identify physical and genetic interactions that implicate E3 identified by differential display (EDD) in promoting spindle assembly checkpoint (SAC) function. During mitosis, the SAC initiates a mitotic checkpoint in response to chromosomes with kinetochores unattached to spindle pole microtubules. Similar to Budding uninhibited by benzimidazoles-related 1 (BUBR1) siRNA, a bona fide SAC component, EDD siRNA abrogated G₂/M accumulation in response to the mitotic destabilizing agent nocodazole. Furthermore, EDD siRNA reduced mitotic cell viability and, in nocodazole-treated cells, increased expression of the promitotic progression protein cell division cycle 20 (CDC20). Copurification studies also identified physical interactions with CDC20, BUBR1, and other components of the SAC. Taken together, these observations highlight the potential role of EDD in regulating mitotic progression and the cellular response to perturbed mitosis.     

Restraining CDK1–cyclin B activation: PP2A on the cUSP(7)

USP7 inhibitors are gaining momentum as a therapeutic strategy to stabilize p53 through their ability to induce MDM2 degradation. However, these inhibitors come with an unexpected p53‐independent toxicity, via an unknown mechanism. In this issue of The EMBO Journal, Galarreta et al report how inhibition of USP7 leads to re‐distribution of PP2A from cytoplasm to nucleus and an increase of deleterious CDK1‐dependent phosphorylation throughout the cell cycle, revealing a new regulatory mechanism for the progression of S‐phase cells toward mitosis to maintain genomic integrity.Subject Categories: Cell Cycle, Post-translational Modifications, Proteolysis & Proteomics

Recent work reveals untimely activation of mitotic cyclin‐dependent kinase as a molecular basis for p53‐independent cell toxicity of USP7 deubiquitinase inhibitors.

The G2‐M transition in the eukaryotic cell cycle is a critical point to ensure that cells with damaged DNA are unable to enter the mitotic phase. This checkpoint is highly regulated by a number of kinases, including ATR, ATM and WEE1, and ends upon activation of the CDK1–cyclin B1 kinase complex (Visconti et al, 2016). Since premature activation of CDK1–cyclin B1 causes replication fork collapse, DNA damage, apoptosis, and mitotic catastrophe (Szmyd et al, 2019 and references therein), restricting CDK1–cyclin B1 activity prior to mitosis is key to maintaining genomic integrity.A body of recent work has suggested that the deubiquitinase USP7 is a master regulator of genomic integrity; it is required for DNA replication in numerous ways, including indirect regulation of cyclin A2 during the S‐phase, origin firing, and replication fork progression. USP7 also regulates mitotic entry by stabilizing PLK1, another kinase which is highly active in the M phase and ensures proper alignment of chromatids prior to segregation. Notably, USP7 inhibitors have become an attractive cancer therapeutic strategy based on their ability to trigger degradation of MDM2, and thereby stabilize p53 (Valles et al, 2020). However, there is growing evidence of USP7 inhibitor‐related toxicity that is not mediated through p53 (Lecona et al, 2016; Agathanggelou et al, 2017), indicating that USP7 inhibitors impact other cellular processes. Therefore, Galarreta et al (2021) investigated the potential functional relationship between USP7 and CDK1, given the role of both factors in regulating the cell cycle.Through a series of in vitro experiments, the authors confirmed that five USP7 inhibitors induce premature mitotic kinase activity, including increased MPM2 signal (indicative of mitosis‐specific phosphorylation events) and phosphorylation of histone H3 Ser10 (H3S10P) in all cells, regardless of where they are in the cell cycle. To determine whether USP7 affects CDK1 during the cell cycle, Galarreta et al (2021) demonstrate that cell lines treated with USP7 inhibitors exhibit reduced levels of inhibitory Tyr‐15 phosphorylation on CDK1 and increased cyclin B1 presence in the nucleus, suggesting activation of the CDK1–cyclin B1 complex. Furthermore, treatment with the CDK1 inhibitor RO3306 rescues the USP7 inhibitor‐dependent increase of mitotic activity.These observations suggest that CDK1 has the potential to catalyze mitosis‐specific phosphorylation irrespective of cell cycle phase and that cells rely on USP7‐specific deubiquitination to suppress or reverse premature CDK1 activity. Surprisingly, despite the nuclear localization of cyclin B and decrease in inhibitory CDK1 Tyr‐15 phosphorylation, USP7 inhibitors failed to drive cells into mitosis. How might this be? Nuclear localization of cyclin B normally occurs just minutes before the onset of mitosis and nuclear envelope breakdown (Santos et al, 2012), yet the nucleus remains intact following USP7 inhibition. Moreover, the decrease in Tyr‐15 phosphorylation suggests the ATR‐ and WEE1‐dependent G2/M checkpoint is inactivated by USP7 inhibition. Do these data hint at the presence of an additional, unknown regulatory mechanism controlling mitotic entry independent of the G2/M checkpoint and nuclear localization of the CDK1–cyclin B complex?To determine whether CDK1 is the driver of USP7 inhibitor toxicity, Galarreta et al exposed cells to CDK1 inhibitors and observed a reduction in apoptosis. Furthermore, CDK1 inhibitors promote cell survival in cells treated with three structurally unrelated USP7 inhibitors. Finally, CDC25A‐deficient mouse embryonic stem cells, which constitutively express low levels of CDK1, resist USP7 inhibition. Together, these data suggest that the USP7 inhibitor‐dependent toxicity is the result of CDK1‐mediated cell death. The authors note that the phosphatase PP2A is an antagonist for CDK1 in addition to being a candidate USP7 substrate (Lecona et al, 2016; Wlodarchak & Xing, 2016), and thus, they turned their attention to elucidating the connection between USP7 and PP2A. Combining biochemical and immunofluorescence studies, Galarreta et al (2021) demonstrate that USP7 interacts with two subunits of PP2A, and this interaction increases in response to USP7 inhibition. Inhibiting USP7 furthermore triggers PP2A re‐localization from the cytoplasm to the nucleus and increases the phosphorylation levels of PP2A substrates, such as AKT and PRC1. DT‐061, a chemical activator of PP2A, reduces CDK1 phosphorylation events, suggesting that PP2A deregulation is a key mediator of USP7 inhibitor‐related toxicity. Using phosphoproteomics to analyze cells treated with a USP7 inhibitor or PP2A‐inhibiting okadaic acid, the authors reveal that both treatments share a significant number of altered phosphorylated targets—especially those related to mitosis, the cell cycle, and epitopes with a CDK‐dependent motif. Thus, the effects of USP7 inhibitors on CDK1 appear to be mediated through PP2A localization to the nucleus.These unexpected findings raise several questions that potentially impact the current view of cell cycle regulation. For example, how does USP7 regulate PP2A localization and is this important for reversing CDK1‐dependent phosphorylation of mitotic substrates prior to mitosis? Does PP2A accumulation in the nucleus explain the failure of USP7‐inhibited cells to enter mitosis despite cyclin B1 nuclear localization? A role for ubiquitin signaling as a regulator of CDK1 in interphase cells has not been reported, and accordingly, new investigations will be needed to unravel the mechanisms by which USP7 controls PP2A localization.Another important question that arises is whether or not CDK1 has sufficient basal activity to phosphorylate numerous mitotic proteins independent of cell cycle phase. The observation that USP7 and PP2A act to prevent the improper accumulation of CDK1‐dependent phosphorylation even in G1 phase cells suggests this to be the case. Alternatively, USP7 activity may be required to prevent abnormal pairing of CDK1 with a cyclin that is ubiquitously expressed across the cell cycle. If so, more research will be needed to uncover how ubiquitin signaling ensures CDK1 only pairs with cyclin A and cyclin B once they accumulate later in the cell cycle.Interestingly, USP7 inhibition also causes a rapid loss in DNA synthesis of S‐phase cells, prompting the authors to perform a time course experiment to decipher the order of events following treatment (i.e., does CDK1 activation precede or follow termination of DNA replication?). High‐throughput microscopy and flow cytometry analysis reveal an immediate reduction of DNA replication, an increase of CDK1 activity, and elevated DNA damage before a detectable increase in H3S10P. Long‐term exposure of USP7 inhibitors leads to DNA damage restricted only to cells with corresponding high levels of H3S10P and MPM2. Overall, these results illustrate how inhibition of USP7 activates CDK1, disrupting DNA replication and inducing DNA damage (Fig 1).Open in a separate windowFigure 1USP7 regulates CDK1In untreated cells, CDK1 is suppressed by USP7 and PP2A, and CDK1‐cyclin B is only active during the G2/M transition. In response to treatment, USP7 facilitates PP2A localization to the nucleus. This allows CDK1 to initiate premature mitotic activity throughout the cell cycle, resulting in increased DNA damage and cellular toxicity.The finding that USP7 inhibitors caused a rapid shutdown of DNA replication brings to mind the recent findings by several groups, that CDK1 activation occurs concomitantly with the S/G2 transition and that premature CDK1 activation in S‐phase terminates replication (Akopyan et al, 2014; Lemmens et al, 2018; Saldivar et al, 2018; Deng et al, 2019; Branigan et al, 2021). According to these studies, coupling of CDK1 activation to the S/G2 transition is regulated by ATR‐CHK1 signaling, a pathway activated by DNA replication to restrain CDK1 through Tyr‐15 phosphorylation. Galarreta et al''s observation that USP7 inhibition overrides ATR‐CHK1 (i.e., Tyr‐15 phosphorylation) highlights the fundamental importance of ubiquitin signaling, and potentially PP2A localization, for ensuring proper S‐to‐M progression and genome maintenance. Ultimately, the mechanistic details of Galarreta et al''s observations remain to be elucidated, and undoubtedly, their findings will inspire future investigations. Moreover, their discovery may lead to a new strategy targeting CDK1 to mitigate unwanted toxicities in the clinic.     

Outcome of treatment of human HeLa cervical cancer cells with roscovitine strongly depends on the dosage and cell cycle status prior to the treatment

Exposure of asynchronously growing human HeLa cervical carcinoma cells to roscovitine (ROSC), a selective cyclin‐dependent kinases (CDKs) inhibitor, arrests their progression at the transition between G₂/M and/or induces apoptosis. The outcome depends on the ROSC concentration. At higher dose ROSC represses HPV‐encoded E7 oncoprotein and initiates caspase‐dependent apoptosis. Inhibition of the site‐specific phosphorylation of survivin and Bad, occurring at high‐dose ROSC treatment, precedes the onset of apoptosis and seems to be a prerequisite for cell death. Considering the fact that in HeLa cells the G₁/S restriction checkpoint is abolished by E7, we addressed the question whether the inhibition of CDKs by pharmacological inhibitors in synchronized cells would be able to block the cell‐cycle in G₁ phase. For this purpose, we attempted to synchronize cells by serum withdrawal or by blocking of the mitotic apparatus using nocodazole. Unlike human MCF‐7 cells, HeLa cells do not undergo G₁ block after serum starvation, but respond with a slight increase of the ratio of G₁ population. Exposure of G₁‐enriched HeLa cells to ROSC after re‐feeding does not block their cell‐cycle progression at G₁‐phase, but increases the ratio of S‐ and G₂‐phase, thereby mimicking the effect on asynchronously growing cells. A quite different impact is observed after treatment of HeLa cells released from mitotic block. ROSC prevents their cell cycle progression and cells transiently accumulate in G₁‐phase. These results show that inhibition of CDKs by ROSC in cells lacking the G₁/S restriction checkpoint has different outcomes depending on the cell‐cycle status prior to the onset of treatment. J. Cell. Biochem. 106: 937–955, 2009. © 2009 Wiley‐Liss, Inc.     

PP2ACdc55 regulates G1 cyclin stability

Maintaining accurate progression through the cell cycle requires the proper temporal expression and regulation of cyclins. The mammalian D-type cyclins promote G₁-S transition. D1 cyclin protein stability is regulated through its ubiquitylation and resulting proteolysis catalyzed by the SCF E3 ubiquitin ligase complex containing the F-box protein, Fbx4. SCF E3-ligase-dependent ubiquitylation of D1 is trigged by an increase in the phosphorylation status of the cyclin. As inhibition of ubiquitin-dependent D1 degradation is seen in many human cancers, we set out to uncover how D-type cyclin phosphorylation is regulated. Here we show that in S. cerevisiae, a heterotrimeric protein phosphatase 2A (PP2A^Cdc55) containing the mammalian PPP2R2/PR55 B subunit ortholog Cdc55 regulates the stability of the G₁ cyclin Cln2 by directly regulating its phosphorylation state. Cells lacking Cdc55 contain drastically reduced Cln2 levels caused by degradation due to cdk-dependent hyperphosphorylation, as a Cln2 mutant unable to be phosphorylated by the yeast cdk Cdc28 is highly stable in cdc55-null cells. Moreover, cdc55-null cells become inviable when the SCF^Grr1 activity known to regulate Cln2 levels is eliminated or when Cln2 is overexpressed, indicating a critical relationship between SCF and PP2A functions in regulating cell cycle progression through modulation of G₁-S cyclin degradation/stability. In sum, our results indicate that PP2A is absolutely required to maintain G₁-S cyclin levels through modulating their phosphorylation status, an event necessary to properly transit through the cell cycle.     

Cutting edge: Chk1 directs senescence and mitotic catastrophe in recovery from G2 checkpoint arrest

Besides the well‐understood DNA damage response via establishment of G₂ checkpoint arrest, novel studies focus on the recovery from arrest by checkpoint override to monitor cell cycle re‐entry. The aim of this study was to investigate the role of Chk1 in the recovery from G₂ checkpoint arrest in HCT116 (human colorectal cancer) wt, p53^–/– and p21^–/– cell lines following H₂O₂ treatment. Firstly, DNA damage caused G₂ checkpoint activation via Chk1. Secondly, overriding G₂ checkpoint led to (i) mitotic slippage, cell cycle re‐entry in G₁ and subsequent G₁ arrest associated with senescence or (ii) premature mitotic entry in the absence of p53/p21^WAF1 causing mitotic catastrophe. We revealed subtle differences in the initial Chk1‐involved G₂ arrest with respect to p53/p21^WAF1: absence of either protein led to late G₂ arrest instead of the classic G₂ arrest during checkpoint initiation, and this impacted the release back into the cell cycle. Thus, G₂ arrest correlated with downstream senescence, but late G₂ arrest led to mitotic catastrophe, although both cell cycle re‐entries were linked to upstream Chk1 signalling. Chk1 knockdown deciphered that Chk1 defines long‐term DNA damage responses causing cell cycle re‐entry. We propose that recovery from oxidative DNA damage‐induced G₂ arrest requires Chk1. It works as cutting edge and navigates cells to senescence or mitotic catastrophe. The decision, however, seems to depend on p53/p21^WAF1. The general relevance of Chk1 as an important determinant of recovery from G₂ checkpoint arrest was verified in HT29 colorectal cancer cells.     

Fructokinase is required for carbon partitioning to cellulose in aspen wood

DNA damage checkpoints delay mitotic cell‐cycle progression in response to DNA stress, stalling the cell cycle to allow time for repair. CDKB is a plant‐specific cyclin‐dependent kinase (CDK) that is required for the G₂/M transition of the cell cycle. In Arabidopsis, DNA damage leads the degradation of CDKB2, and the subsequent G₂ arrest gives cells time to repair damaged DNA. G₂ arrest also triggers transition from the mitotic cycle to endoreduplication, leading to the presence of polyploid cells in many tissues. In contrast, in rice (Oryza sativa), polyploid cells are found only in the endosperm. It was unclear whether endoreduplication contributes to alleviating DNA damage in rice (Oryza sativa). Here, we show that DNA damage neither down‐regulates Orysa;CDKB2;1 nor induces endoreduplication in rice. Furthermore, we found increased levels of Orysa;CDKB2;1 protein upon DNA damage. These results suggest that CDKB2 functions differently in Arabidopsis and rice in response to DNA damage. Arabidopsis may adopt endoreduplication as a survival strategy under genotoxic stress conditions, but rice may enhance DNA repair capacity upon genotoxic stress. In addition, polyploid cells due to endomitosis were present in CDKB2;1 knockdown rice, suggesting an important role for Orysa;CDKB2;1 during mitosis.     

Regulatory dephosphorylation of CDK at G₂/M in plants: yeast mitotic phosphatase cdc25 induces cytokinin-like effects in transgenic tobacco morphogenesis

Background

During the last three decades, the cell cycle and its control by cyclin-dependent kinases (CDKs) have been extensively studied in eukaryotes. This endeavour has produced an overall picture that basic mechanisms seem to be largely conserved among all eukaryotes. The intricate regulation of CDK activities includes, among others, CDK activation by CDC25 phosphatase at G₂/M. In plants, however, studies of this regulation have lagged behind as a plant Cdc25 homologue or other unrelated phosphatase active at G₂/M have not yet been identified.

Scope

Failure to identify a plant mitotic CDK activatory phosphatase led to characterization of the effects of alien cdc25 gene expression in plants. Tobacco, expressing the Schizosaccharomyces pombe mitotic activator gene, Spcdc25, exhibited morphological, developmental and biochemical changes when compared with wild type (WT) and, importantly, increased CDK dephosphorylation at G₂/M. Besides changes in leaf shape, internode length and root development, in day-neutral tobacco there was dramatically earlier onset of flowering with a disturbed acropetal floral capacity gradient typical of WT. In vitro, de novo organ formation revealed substantially earlier and more abundant formation of shoot primordia on Spcdc25 tobacco stem segments grown on shoot-inducing media when compared with WT. Moreover, in contrast to WT, stem segments from transgenic plants formed shoots even without application of exogenous growth regulator. Spcdc25-expressing BY-2 cells exhibited a reduced mitotic cell size due to a shortening of the G₂ phase together with high activity of cyclin-dependent kinase, NtCDKB1, in early S-phase, S/G₂ and early M-phase. Spcdc25-expressing tobacco (‘Samsun’) cell suspension cultures showed a clustered, more circular, cell phenotype compared with chains of elongated WT cells, and increased content of starch and soluble sugars. Taken together, Spcdc25 expression had cytokinin-like effects on the characteristics studied, although determination of endogenous cytokinin levels revealed a dramatic decrease in Spcdc25 transgenics.

Conclusions

The data gained using the plants expressing yeast mitotic activator, Spcdc25, clearly argue for the existence and importance of activatory dephosphorylation at G₂/M transition and its interaction with cytokinin signalling in plants. The observed cytokinin-like effects of Spcdc25 expression are consistent with the concept of interaction between cell cycle regulators and phytohormones during plant development. The G₂/M control of the plant cell cycle, however, remains an elusive issue as doubts persist about the mode of activatory dephosphorylation, which in other eukaryotes is provided by Cdc25 phosphatase serving as a final all-or-nothing mitosis regulator.