A new negative feedback mechanism for MAPK pathway inactivation through Srk1 MAPKAP kinase.

The fission yeast mitogen-activated kinase (MAPK) Sty1 is essential for cell survival in response to different environmental insults. In unstimulated cells, Sty1 forms an inactive ternary cytoplasmatic complex with the MAPKK Wis1 and the MAPKAP kinase Srk1. Wis1 phosphorylates and activates Sty1, inducing the nuclear translocation of the complex. Once in the nucleus, Sty1 phosphorylates and activates Srk1, which in turns inhibits Cdc25 and cell cycle progression, before being degraded in a proteasome-dependent manner. In parallel, active nuclear Sty1 activates the transcription factor Atf1, which results in the expression of stress response genes including pyp2 (a MAPK phosphatase) and srk1. Despite its essentiality in response to stress, persistent activation of the MAPK pathway can be deleterious and induces cell death. Thus, timely pathway inactivation is essential to ensure an appropriate response and cell viability. Here, uncover a role for the MAPKAP kinase Srk1 as an essential component of a negative feedback loop regulating the Sty1 pathway through phosphorylation and inhibition of the Wis1 MAPKK. This feedback regulation by a downstream kinase in the pathway highlights an additional mechanism for fine-tuning of MAPK signaling. Thus, our results indicate that Srk1 not only facilitates the adaptation to stress conditions by preventing cell cycle progression, but also plays an instrumental role regulating the upstream kinases in the stress MAPK pathway.

Uncoupling of Mitosis and Cytokinesis Upon a Prolonged Arrest in Metaphase Is Influenced by Protein Phosphatases and Mitotic Transcription in Fission Yeast.

Depletion of the Anaphase-Promoting Complex/Cyclosome (APC/C) activator Cdc20 arrests cells in metaphase with high levels of the mitotic cyclin (Cyclin B) and the Separase inhibitor Securin. In mammalian cells this arrest has been exploited for the treatment of cancer with drugs that engage the spindle assembly checkpoint and, recently, with chemical inhibitors of the APC/C. While most cells arrested in mitosis for prolonged periods undergo apoptosis, others skip cytokinesis and enter G1 with unsegregated chromosomes. This process, known as mitotic slippage, generates aneuploidy and increases genomic instability in the cancer cell. Here, we analyze the behavior of fission yeast cells arrested in mitosis through the transcriptional silencing of the Cdc20 homolog slp1. While depletion of slp1 readily halts cells in metaphase, this arrest is only transient and a majority of cells eventually undergo cytokinesis and show steady mitotic dephosphorylation. Notably, this occurs in the absence of Cyclin B (Cdc13) degradation. We investigate the involvement of phosphatase activity in these events and demonstrate that PP2A-B55Pab1 is required to prevent septation and, during the arrest, its CDK-mediated inhibition facilitates the induction of cytokinesis. In contrast, deletion of PP2A-B56Par1 completely abrogates septation. We show that this effect is partly due to this mutant entering mitosis with reduced CDK activity. Interestingly, both PP2A-B55Pab1 and PP2A-B56Par1, as well as Clp1 (the homolog of the budding yeast mitotic phosphatase Cdc14) are required for the dephosphorylation of mitotic substrates during the escape. Finally, we show that the mitotic transcriptional wave controlled by the RFX transcription factor Sak1 facilitates the induction of cytokinesis and also requires the activity of PP2A-B56Par1 in a mechanism independent of CDK.

Requirement of PP2A-B56Par1 for the Stabilization of the CDK Inhibitor Rum1 and Activation of APC/CSte9 during Pre-Start G1 in S. pombe.

Exit from the cell cycle during the establishment of quiescence and upon cell differentiation requires the sustained inactivation of CDK complexes. Fission yeast cells deprived of nitrogen halt cell cycle progression in pre-Start G1, before becoming quiescent or undergoing sexual differentiation. The CDK inhibitor Rum1 and the APC/C activator Ste9 are fundamental for this arrest, but both are down-regulated by CDK complexes. Here, we show that PP2A-B56Par1 is instrumental for Rum1 stabilization and Ste9 activation. In the absence of PP2A-B56Par1, cells fail to accumulate Rum1, and this results in persistent CDK activity, Ste9 inactivation, retention of the mitotic cyclin Cdc13, and impaired withdrawal from the cell cycle during nitrogen starvation. Importantly, mutation of a putative B56 interacting motif in Rum1 recapitulates these defects. These results underscore the relevance of CDK-counteracting phosphatases in cell differentiation, establishment of the quiescent state, and escape from it in cancer cells.

Protein Phosphatases in G1 Regulation.

Eukaryotic cells make the decision to proliferate, to differentiate or to cease dividing during G1, before passage through the restriction point or Start. Keeping cyclin-dependent kinase (CDK) activity low during this period restricts commitment to a new cell cycle and is essential to provide the adequate timeframe for the sensing of environmental signals. Here, we review the role of protein phosphatases in the modulation of CDK activity and as the counteracting force for CDK-dependent substrate phosphorylation, in budding and fission yeast. Moreover, we discuss recent findings that place protein phosphatases in the interface between nutritional signalling pathways and the cell cycle machinery.

High Throughput Chemical Screening Reveals Multiple Regulatory Proteins on FOXA1 in Breast Cancer Cell Lines.

Forkhead box A1 (FOXA1) belongs to the forkhead class transcription factor family, playing pioneering function for hormone receptors in breast and prostate cancers, and mediating activation of linage specific enhancers. Interplay between FOXA1 and breast cancer specific signaling pathways has been reported previously, indicating a regulation network on FOXA1 in breast cancer cells. Here in this study, we aimed to identify which are the proteins that could potentially control FOXA1 function in breast cancer cell lines expressing different molecular markers. We first established a luciferase reporter system reflecting FOXA1 binding to DNA. Then, we applied high throughput chemical screening of multiple protein targets and mass spectrometry in breast cancer cell lines expressing different molecular markers: ER positive/HER2 negative (MCF-7), ER positive/HER2 positive (BT474), and ER negative/HER2 positive (MDA-MB-453). Regardless of estrogen receptor status, HER2 (human epidermal growth factor receptor 2) enriched cell lines showed similar response to kinase inhibitors, indicating the control of FOXA1 by cell signaling kinases. Among these kinases, we identified additional receptor tyrosine kinases and cyclin-dependent kinases as regulators of FOXA1. Furthermore, we performed proteomics experiments from FOXA1 inmunoprecipitated protein complex to identify that FOXA1 interacts with several proteins. Among all the targets, we identified cyclin-dependent kinase 1 (CDK1) as a positive factor to interact with FOXA1 in BT474 cell line. In silico analyses confirmed that cyclin-dependent kinases might be the kinases responsible for FOXA1 phosphorylation at the Forkhead domain and the transactivation domain. These results reveal that FOXA1 is potentially regulated by multiple kinases. The cell cycle control kinase CDK1 might control directly FOXA1 by phosphorylation and other kinases indirectly by means of regulating other proteins.

Express yourself: how PP2A-B55Pab1 helps TORC1 talk to TORC2.

The control of cell fate, growth and proliferation in response to nitrogen availability is a tightly controlled process, with the two TOR complexes (TORC1 and TORC2) and their effectors playing a central role. PP2A-B55Pab1 has recently been shown to be a key element in this response in fission yeast, where it regulates cell cycle progression and sexual differentiation. Importantly, a recent study from our group has shown that PP2A-B55Pab1 acts as a mediator between the activities of the two TOR signaling modules, enabling a crosstalk that is required to engage in the differentiation program. In this review, we recapitulate the studies that have led to our current understanding of the interplay between TOR complexes. Moreover, we discuss several aspects of the response to nitrogen availability that still require further attention, and which will be important in the future to fully realize the implications of phosphatase activity in the context of TOR signaling.

A checkpoint-independent mechanism delays entry into mitosis after UV irradiation.

When cells are exposed to stress they delay entry into mitosis. The most extensively studied mechanism behind this delay is the DNA-damage-induced G2/M checkpoint. Here, we show the existence of an additional stress-response pathway in Schizosaccharomyces pombe that is independent of the classic ATR/Rad3-dependent checkpoint. This novel mechanism delays entry mitosis independently of the spindle assembly checkpoint and the mitotic kinases Fin1, Ark1 and Plo1. The pathway delays activation of the mitotic cyclin-dependent kinase (CDK) Cdc2 after UV irradiation. Furthermore, we demonstrate that translation of the mitotic cyclin Cdc13 is selectively downregulated after UV irradiation, and we propose that this downregulation of Cdc13 contributes to the delayed activation of Cdc2 and the delayed mitosis.

A PP2A-B55-Mediated Crosstalk between TORC1 and TORC2 Regulates the Differentiation Response in Fission Yeast.

Extracellular cues regulate cell fate, and this is mainly achieved through the engagement of specific transcriptional programs. The TORC1 and TORC2 complexes mediate the integration of nutritional cues to cellular behavior, but their interplay is poorly understood. Here, we use fission yeast to investigate how phosphatase activity participates in this interplay during the switch from proliferation to sexual differentiation. We find that loss of PP2A-B55Pab1 enhances the expression of differentiation-specific genes and leads to premature conjugation. pab1 deletion brings about a transcriptional profile similar to TORC1 inactivation, and deletion of pab1 overcomes the repression of differentiation genes in cells overexpressing TORC1. Importantly, we show that this effect is mediated by an increased TORC2-AKT (Gad8) signaling. Under nutrient-rich conditions, PP2A-B55Pab1 dephosphorylates Gad8 Ser546, repressing its activity. Conversely, TORC1 inactivation upon starvation leads to the inactivation of PP2A-B55Pab1 through the Greatwall-Endosulfin pathway. This results in the activation of Gad8 and the commitment to differentiation. Thus, PP2A-B55Pab1 enables a crosstalk between the two TOR complexes that controls cell-fate decisions in response to nutrient availability.

Ssp1 CaMKK: A Sensor of Actin Polarization That Controls Mitotic Commitment through Srk1 in Schizosaccharomyces pombe.

BACKGROUND: Calcium/calmodulin-dependent protein kinase kinase (CaMKK) is required for diverse cellular functions. Mammalian CaMKK activates CaMKs and also the evolutionarily-conserved AMP-activated protein kinase (AMPK). The fission yeast Schizosaccharomyces pombe CaMKK, Ssp1, is required for tolerance to limited glucose through the AMPK, Ssp2, and for the integration of cell growth and division through the SAD kinase Cdr2. RESULTS: Here we report that Ssp1 controls the G2/M transition by regulating the activity of the CaMK Srk1. We show that inhibition of Cdc25 by Srk1 is regulated by Ssp1; and also that restoring growth polarity and actin localization of ssp1-deleted cells by removing the actin-monomer-binding protein, twinfilin, is sufficient to suppress the ssp1 phenotype. CONCLUSIONS: These findings demonstrate that entry into mitosis is mediated by a network of proteins, including the Ssp1 and Srk1 kinases. Ssp1 connects the network of components that ensures proper polarity and cell size with the network of proteins that regulates Cdk1-cyclin B activity, in which Srk1 plays an inhibitory role.

A quantitative model for cyclin-dependent kinase control of the cell cycle: revisited.

The eukaryotic cell division cycle encompasses an ordered series of events. Chromosomal DNA is replicated during S phase of the cell cycle before being distributed to daughter cells in mitosis. Both S phase and mitosis in turn consist of an intricately ordered sequence of molecular events. How cell cycle ordering is achieved, to promote healthy cell proliferation and avert insults on genomic integrity, has been a theme of Paul Nurse's research. To explain a key aspect of cell cycle ordering, sequential S phase and mitosis, Stern & Nurse proposed 'A quantitative model for cdc2 control of S phase and mitosis in fission yeast'. In this model, S phase and mitosis are ordered by their dependence on increasing levels of cyclin-dependent kinase (Cdk) activity. Alternative mechanisms for ordering have been proposed that rely on checkpoint controls or on sequential waves of cyclins with distinct substrate specificities. Here, we review these ideas in the light of experimental evidence that has meanwhile accumulated. Quantitative Cdk control emerges as the basis for cell cycle ordering, fine-tuned by cyclin specificity and checkpoints. We propose a molecular explanation for quantitative Cdk control, based on thresholds imposed by Cdk-counteracting phosphatases, and discuss its implications.

Cell cycle: the art of multi-tasking.

Separase is the protease that cleaves the cohesive link between sister chromatids to trigger chromosome segregation in mitosis and meiosis. This enzyme is known to orchestrate additional mitotic events and we now gain new insight into how it promotes cytokinesis in the nematode Caenorhabditis elegans.

System-level feedbacks control cell cycle progression.

Repetitive cell cycles, which are essential to the perpetuation of life, are orchestrated by an underlying biochemical reaction network centered around cyclin-dependent protein kinases (Cdks) and their regulatory subunits (cyclins). Oscillations of Cdk1/CycB activity between low and high levels during the cycle trigger DNA replication and mitosis in the correct order. Based on computational modeling, we proposed that the low and the high kinase activity states are alternative stable steady states of a bistable Cdk-control system. Bistability is a consequence of system-level feedback (positive and double-negative feedback signals) in the underlying control system. We have also argued that bistability underlies irreversible transitions between low and high Cdk activity states and thereby ensures directionality of cell cycle progression.

Irreversibility of mitotic exit is the consequence of systems-level feedback.

The eukaryotic cell cycle comprises an ordered series of events, orchestrated by the activity of cyclin-dependent kinases (Cdks), leading from chromosome replication during S phase to their segregation in mitosis. The unidirectionality of cell-cycle transitions is fundamental for the successful completion of this cycle. It is thought that irrevocable proteolytic degradation of key cell-cycle regulators makes cell-cycle transitions irreversible, thereby enforcing directionality. Here we have experimentally examined the contribution of cyclin proteolysis to the irreversibility of mitotic exit, the transition from high mitotic Cdk activity back to low activity in G1. We show that forced cyclin destruction in mitotic budding yeast cells efficiently drives mitotic exit events. However, these remain reversible after termination of cyclin proteolysis, with recovery of the mitotic state and cyclin levels. Mitotic exit becomes irreversible only after longer periods of cyclin degradation, owing to activation of a double-negative feedback loop involving the Cdk inhibitor Sic1 (refs 4, 5). Quantitative modelling suggests that feedback is required to maintain low Cdk activity and to prevent cyclin resynthesis. Our findings demonstrate that the unidirectionality of mitotic exit is not the consequence of proteolysis but of systems-level feedback required to maintain the cell cycle in a new stable state.

Estrogen receptor beta displays cell cycle-dependent expression and regulates the G1 phase through a non-genomic mechanism in prostate carcinoma cells.

BACKGROUND: It is well known that estrogens regulate cell cycle progression, but the specific contributions and mechanisms of action of the estrogen receptor beta (ERbeta) remain elusive. METHODS: We have analyzed the levels of ERbeta1 and ERbeta2 throughout the cell cycle, as well as the mechanisms of action and the consequences of the over-expression of ERbeta1 in the human prostate cancer LNCaP cell line. RESULTS: Both ERbeta1 mRNA and protein expression increased from the G1 to the S phase and decreased before entering the G2/M phase, whereas ERbeta2 levels decreased during the S phase and increased in the G2/M phase. ERbeta1 protein was detected in both the nuclear and non-nuclear fractions, and ERbeta2 was found exclusively in the nucleus. Regarding the mechanisms of action, endogenous ERbeta was able to activate transcription via ERE during the S phase in a ligand-dependent manner, whereas no changes in AP1 and NFkappaB transactivation were observed after exposure to estradiol or the specific inhibitor ICI 182,780. Over-expression of either wild type ERbeta1 or ERbeta1 mutated in the DNA-binding domain caused an arrest in early G1. This arrest was accompanied by the interaction of over-expressed ERbeta1 with c-Jun N-terminal protein kinase 1 (JNK1) and a decrease in c-Jun phosphorylation and cyclin D1 expression. The administration of ICI impeded the JNK1-ERbeta1 interaction, increased c-Jun phosphorylation and cyclin D1 expression and allowed the cells to progress to late G1, where they became arrested. CONCLUSIONS: Our results demonstrate that, in LNCaP prostate cancer cells, both ERbeta isoforms are differentially expressed during the cell cycle and that ERbeta regulates the G1 phase by a non-genomic mechanism.

Crosstalk between Nap1 protein and Cds1 checkpoint kinase to maintain chromatin integrity.

The nucleosome assembly protein Nap1 has been implicated in various cellular functions such as histone shuttling into the nucleus, nucleosome assembly, chromatin remodelling, transcriptional control and cell-cycle regulation in Saccharomyces cerevisiae. In Schizosaccharomyces pombe nap1 null mutant cells are viable but they showed a delay in the onset of mitosis which is rescued by the absence of the replication Cds1 checkpoint kinase. In contrast, the absence of the DNA-damage Chk1 checkpoint kinase is unable to rescue the delay. Moreover, the double nap1 cds1 mutant cells lose viability and cells show positive H2AX phosphorylation, suggesting that the viability of nap1-deleted cells is due to the Cds1 kinase. We also show that overexpression of Nap1 protein blocks the cell cycle in G1 phase.

Activation of Srk1 by the mitogen-activated protein kinase Sty1/Spc1 precedes its dissociation from the kinase and signals its degradation.

Control of cell cycle progression by stress-activated protein kinases (SAPKs) is essential for cell adaptation to extracellular stimuli. The Schizosaccharomyces pombe SAPK Sty1/Spc1 orchestrates general changes in gene expression in response to diverse forms of cytotoxic stress. Here we show that Sty1/Spc1 is bound to its target, the Srk1 kinase, when the signaling pathway is inactive. In response to stress, Sty1/Spc1 phosphorylates Srk1 at threonine 463 of the regulatory domain, inducing both activation of Srk1 kinase, which negatively regulates cell cycle progression by inhibiting Cdc25, and dissociation of Srk1 from the SAPK, which leads to Srk1 degradation by the proteasome.

Inactivation of the Cdc25 phosphatase by the stress-activated Srk1 kinase in fission yeast.

The mechanisms by which environmental stress regulates cell cycle progression are poorly understood. In fission yeast, we show that Srk1 kinase, which associates with the stress-activated p38/Sty1 MAP kinase, regulates the onset of mitosis by inhibiting the Cdc25 phosphatase. Srk1 is periodically active in G2, and its overexpression causes cell cycle arrest in late G2 phase, whereas cells lacking srk1 enter mitosis prematurely. We find that Srk1 interacts with and phosphorylates Cdc25 at the same sites phosphorylated by the Chk1 and Cds1 (Chk2) kinases and that this phosphorylation is necessary for Srk1 to delay mitotic entry. Phosphorylation by Srk1 causes Cdc25 to bind to Rad24, a 14-3-3 protein family member, and accumulation of Cdc25 in the cytoplasm. However, Srk1 does not regulate Cdc25 in response to replication arrest or DNA damage but, rather, during a normal cell cycle and in response to nongenotoxic environmental stress.