MAPK-dependent control of mitotic progression in S. pombe.

BACKGROUND: Mitogen-activated protein kinases (MAPKs) preserve cell homeostasis by transducing physicochemical fluctuations of the environment into multiple adaptive responses. These responses involve transcriptional rewiring and the regulation of cell cycle transitions, among others. However, how stress conditions impinge mitotic progression is largely unknown. The mitotic checkpoint is a surveillance mechanism that inhibits mitotic exit in situations of defective chromosome capture, thus preventing the generation of aneuploidies. In this study, we investigate the role of MAPK Pmk1 in the regulation of mitotic exit upon stress. RESULTS: We show that Schizosaccharomyces pombe cells lacking Pmk1, the MAP kinase effector of the cell integrity pathway (CIP), are hypersensitive to microtubule damage and defective in maintaining a metaphase arrest. Epistasis analysis suggests that Pmk1 is involved in maintaining spindle assembly checkpoint (SAC) signaling, and its deletion is additive to the lack of core SAC components such as Mad2 and Mad3. Strikingly, pmk1Δ cells show up to twofold increased levels of the anaphase-promoting complex (APC/C) activator Cdc20Slp1 during unperturbed growth. We demonstrate that Pmk1 physically interacts with Cdc20Slp1 N-terminus through a canonical MAPK docking site. Most important, the Cdc20Slp1 pool is rapidly degraded in stressed cells undergoing mitosis through a mechanism that requires MAPK activity, Mad3, and the proteasome, thus resulting in a delayed mitotic exit. CONCLUSIONS: Our data reveal a novel function of MAPK in preventing mitotic exit and activation of cytokinesis in response to stress. The regulation of Cdc20Slp1 turnover by MAPK Pmk1 provides a key mechanism by which the timing of mitotic exit can be adjusted relative to environmental conditions.

Loss of UBE2S causes meiosis I arrest with normal spindle assembly checkpoint dynamics in mouse oocytes.

The timely degradation of proteins that regulate the cell cycle is essential for oocyte maturation. Oocytes are equipped to degrade proteins via the ubiquitin-proteasome system. In meiosis, anaphase promoting complex/cyclosome (APC/C), an E3 ubiquitin-ligase, is responsible for the degradation of proteins. Ubiquitin-conjugating enzyme E2 S (UBE2S), an E2 ubiquitin-conjugating enzyme, delivers ubiquitin to APC/C. APC/C has been extensively studied, but the functions of UBE2S in oocyte maturation and mouse fertility are not clear. In this study, we used Ube2s knockout mice to explore the role of UBE2S in mouse oocytes. Ube2s-deleted oocytes were characterized by meiosis I arrest with normal spindle assembly and spindle assembly checkpoint dynamics. However, the absence of UBE2S affected the activity of APC/C. Cyclin B1 and securin are two substrates of APC/C, and their levels were consistently high, resulting in the failure of homologous chromosome separation. Unexpectedly, the oocytes arrested in meiosis I could be fertilized and the embryos could become implanted normally, but died before embryonic day 10.5. In conclusion, our findings reveal an indispensable regulatory role of UBE2S in mouse oocyte meiosis and female fertility.

Genome-Wide Identification and Analysis of APC E3 Ubiquitin Ligase Genes Family in Triticum aestivum.

E3 ubiquitin ligases play a pivotal role in ubiquitination, a crucial post-translational modification process. Anaphase-promoting complex (APC), a large cullin-RING E3 ubiquitin ligase, regulates the unidirectional progression of the cell cycle by ubiquitinating specific target proteins and triggering plant immune responses. Several E3 ubiquitin ligases have been identified owing to advancements in sequencing and annotation of the wheat genome. However, the types and functions of APC E3 ubiquitin ligases in wheat have not been reported. This study identified 14 members of the APC gene family in the wheat genome and divided them into three subgroups (CCS52B, CCS52A, and CDC20) to better understand their functions. Promoter sequence analysis revealed the presence of several cis-acting elements related to hormone and stress responses in the APC E3 ubiquitin ligases in wheat. All identified APC E3 ubiquitin ligase family members were highly expressed in the leaves, and the expression of most genes was induced by the application of methyl jasmonate (MeJA). In addition, the APC gene family in wheat may play a role in plant defense mechanisms. This study comprehensively analyzes APC genes in wheat, laying the groundwork for future research on the function of APC genes in response to viral infections and expanding our understanding of wheat immunity mechanisms.

Functional analysis of Cdc20 reveals a critical role of CRY box in mitotic checkpoint signaling.

Accurate mitosis is coordinated by the spindle assembly checkpoint (SAC) through the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex or cyclosome (APC/C). As an essential regulator, Cdc20 promotes mitotic exit through activating APC/C and monitors kinetochore-microtubule attachment through activating SAC. Cdc20 requires multiple interactions with APC/C and MCC subunits to elicit these functions. Functionally assessing these interactions within cells requires efficient depletion of endogenous Cdc20, which is highly difficult to achieve by RNA interference (RNAi). Here we generated Cdc20 RNAi-sensitive cell lines which display a penetrant metaphase arrest by a single RNAi treatment. In this null background, we accurately measured the contribution of each known motif of Cdc20 on APC/C and SAC activation. The CRY box, a previously identified degron, was found critical for SAC by promoting MCC formation and its interaction with APC/C. These data reveal additional regulation within the SAC and establish a novel method to interrogate Cdc20.

Weakened APC/C activity at mitotic exit drives cancer vulnerability to KIF18A inhibition.

The efficacy of current antimitotic cancer drugs is limited by toxicity in highly proliferative healthy tissues. A cancer-specific dependency on the microtubule motor protein KIF18A therefore makes it an attractive therapeutic target. Not all cancers require KIF18A, however, and the determinants underlying this distinction remain unclear. Here, we show that KIF18A inhibition drives a modest and widespread increase in spindle assembly checkpoint (SAC) signaling from kinetochores which can result in lethal mitotic delays. Whether cells arrest in mitosis depends on the robustness of the metaphase-to-anaphase transition, and cells predisposed with weak basal anaphase-promoting complex/cyclosome (APC/C) activity and/or persistent SAC signaling through metaphase are uniquely sensitive to KIF18A inhibition. KIF18A-dependent cancer cells exhibit hallmarks of this SAC:APC/C imbalance, including a long metaphase-to-anaphase transition, and slow mitosis overall. Together, our data reveal vulnerabilities in the cell division apparatus of cancer cells that can be exploited for therapeutic benefit.

ZmTDM1 encodes a tetratricopeptide repeat domain protein and is required for meiotic exit in maize.

Elaborate cell-cycle control must be adopted to ensure the continuity of the meiotic second division and termination after that. Despite its importance, however, the genetic controls underlying the meiotic cell cycle have not been reported in maize. Here, we characterized a meiotic cell-cycle controller ZmTDM1, which is a homolog of Arabidopsis TDM1 and encodes a canonical tetratricopeptide repeat domain protein in maize. The Zmtdm1 homozygous plants exhibited complete male sterility and severe female abortion. In Zmtdm1 mutants, cell-cycle progression was almost identical to that of wild type from leptotene to anaphase II. However, chromosomes in the tetrad failed meiotic termination at the end of the second division and underwent additional divisions in succession without DNA replication, reducing the ploidy to less than haploid in the product. In addition, two ZmTDM1-like homologs (ZmTDML1 and ZmTDML2) were not functional in meiotic cell-cycle control. Moreover, ZmTDM1 interacted with RING-type E3 ubiquitin ligase, revealing that it acts as a subunit of the APC/C E3 ubiquitin ligase complex. Overall, our results identified a regulator of meiotic cell cycle in maize and demonstrated that ZmTDM1 is essential for meiotic exit after meiosis II.

SETDB2 interacts with BUBR1 to induce accurate chromosome segregation independently of its histone methyltransferase activity.

SETDB2 is a H3K9 histone methyltransferase required for accurate chromosome segregation. Its H3K9 histone methyltransferase activity was reported to be associated with chromosomes during metaphase. Here, we confirm that SETDB2 is required for mitosis and accurate chromosome segregation. However, these functions are independent of its histone methyltransferase activity. Further analysis showed that SETDB2 can interact with BUBR1, and is required for CDC20 binding to BUBR1 and APC/C complex and CYCLIN B1 degradation. The ability of SETDB2 to regulate the binding of CDC20 to BUBR1 or APC/C complex, and stabilization of CYCLIN B1 are also independent of its histone methyltransferase activity. These results suggest that SETDB2 interacts with BUBR1 to promote binding of CDC20 to BUBR1 and APC3, then degrades CYCLIN B1 to ensure accurate chromosome segregation and mitosis, independently of its histone methyltransferase activity.

TUBA1A licenses APC/C-mediated mitotic progression to drive glioblastoma growth by inhibiting PLK3.

Glioblastoma (GBM) is the most common, aggressive, and chemorefractory primary brain tumor in adults. Identifying novel drug targets is crucial for GBM treatment. Here, we demonstrate that tubulin alpha 1a (TUBA1A) is significantly upregulated in GBM compared to low-grade gliomas (LGG) and normal tissues. High TUBA1A expression is associated with poor survival in GBM patients. TUBA1A knockdown results in mitotic arrest and reduces tumor growth in mice. TUBA1A interacts with the polo-like kinase 3 (PLK3) in the cytoplasm to inhibit its activation. This interaction licenses activation of the anaphase-promoting complex or cyclosome (APC/C) to ensure proper Foxm1-mediated metaphase-to-anaphase transition and mitotic exit. Overall, our findings demonstrate that targeting TUBA1A attenuates GBM cell growth by suppressing mitotic progression in a PLK3-dependent manner.

Spo13/MEIKIN ensures a Two-Division meiosis by preventing the activation of APC/CAma1 at meiosis I.

Genome haploidization at meiosis depends on two consecutive nuclear divisions, which are controlled by an oscillatory system consisting of Cdk1-cyclin B and the APC/C bound to the Cdc20 activator. How the oscillator generates exactly two divisions has been unclear. We have studied this question in yeast where exit from meiosis involves accumulation of the APC/C activator Ama1 at meiosis II. We show that inactivation of the meiosis I-specific protein Spo13/MEIKIN results in a single-division meiosis due to premature activation of APC/CAma1 . In the wild type, Spo13 bound to the polo-like kinase Cdc5 prevents Ama1 synthesis at meiosis I by stabilizing the translational repressor Rim4. In addition, Cdc5-Spo13 inhibits the activity of Ama1 by converting the B-type cyclin Clb1 from a substrate to an inhibitor of Ama1. Cdc20-dependent degradation of Spo13 at anaphase I unleashes a feedback loop that increases Ama1's synthesis and activity, leading to irreversible exit from meiosis at the second division. Thus, by repressing the exit machinery at meiosis I, Cdc5-Spo13 ensures that cells undergo two divisions to produce haploid gametes.

Meiosis II spindle disassembly requires two distinct pathways.

During exit from meiosis II, cells undergo several structural rearrangements, including disassembly of the meiosis II spindles and cytokinesis. Each of these changes is regulated to ensure that they occur at the proper time. Previous studies have demonstrated that both SPS1, which encodes a STE20-family GCKIII kinase, and AMA1, which encodes a meiosis-specific activator of the Anaphase Promoting Complex, are required for both meiosis II spindle disassembly and cytokinesis in the budding yeast Saccharomyces cerevisiae. We examine the relationship between meiosis II spindle disassembly and cytokinesis and find that the meiosis II spindle disassembly failure in sps1Δ and ama1∆ cells is not the cause of the cytokinesis defect. We also see that the spindle disassembly defects in sps1Δ and ama1∆ cells are phenotypically distinct. We examined known microtubule-associated proteins Ase1, Cin8, and Bim1, and found that AMA1 is required for the proper loss of Ase1 and Cin8 on meiosis II spindles while SPS1 is required for Bim1 loss in meiosis II. Taken together, these data indicate that SPS1 and AMA1 promote distinct aspects of meiosis II spindle disassembly, and that both pathways are required for the successful completion of meiosis.

SIRT1 ubiquitination is regulated by opposing activities of APC/C-Cdh1 and AROS during stress-induced premature senescence.

SIRT1, a member of the mammalian sirtuin family, is a nicotinamide adenosine dinucleotide (NAD)-dependent deacetylase with key roles in aging-related diseases and cellular senescence. However, the mechanism by which SIRT1 protein homeostasis is controlled under senescent conditions remains elusive. Here, we revealed that SIRT1 protein is significantly downregulated due to ubiquitin-mediated proteasomal degradation during stress-induced premature senescence (SIPS) and that SIRT1 physically associates with anaphase-promoting complex/cyclosome (APC/C), a multisubunit E3 ubiquitin ligase. Ubiquitin-dependent SIRT1 degradation is stimulated by the APC/C coactivator Cdh1 and not by the coactivator Cdc20. We found that Cdh1 depletion impaired the SIPS-promoted downregulation of SIRT1 expression and reduced cellular senescence, likely through SIRT1-driven p53 inactivation. In contrast, AROS, a SIRT1 activator, reversed the SIRT1 degradation induced by diverse stressors and antagonized Cdh1 function through competitive interactions with SIRT1. Furthermore, our data indicate opposite roles for Cdh1 and AROS in the epigenetic regulation of the senescence-associated secretory phenotype genes IL-6 and IL-8. Finally, we demonstrated that pinosylvin restores downregulated AROS (and SIRT1) expression levels in bleomycin-induced mouse pulmonary senescent tissue while repressing bleomycin-promoted Cdh1 expression. Overall, our study provides the first evidence of the reciprocal regulation of SIRT1 stability by APC/C-Cdh1 and AROS during stress-induced premature senescence, and our findings suggest pinosylvin as a potential senolytic agent for pulmonary fibrosis.

Multiple intermolecular interactions facilitate rapid evolution of essential genes.

Essential genes are commonly assumed to function in basic cellular processes and to change slowly. However, it remains unclear whether all essential genes are similarly conserved or if their evolutionary rates can be accelerated by specific factors. To address these questions, we replaced 86 essential genes of Saccharomyces cerevisiae with orthologues from four other species that diverged from S. cerevisiae about 50, 100, 270 and 420 Myr ago. We identify a group of fast-evolving genes that often encode subunits of large protein complexes, including anaphase-promoting complex/cyclosome (APC/C). Incompatibility of fast-evolving genes is rescued by simultaneously replacing interacting components, suggesting it is caused by protein co-evolution. Detailed investigation of APC/C further revealed that co-evolution involves not only primary interacting proteins but also secondary ones, suggesting the evolutionary impact of epistasis. Multiple intermolecular interactions in protein complexes may provide a microenvironment facilitating rapid evolution of their subunits.

Principles and dynamics of spindle assembly checkpoint signalling.

The transmission of a complete set of chromosomes to daughter cells during cell division is vital for development and tissue homeostasis. The spindle assembly checkpoint (SAC) ensures correct segregation by informing the cell cycle machinery of potential errors in the interactions of chromosomes with spindle microtubules prior to anaphase. To do so, the SAC monitors microtubule engagement by specialized structures known as kinetochores and integrates local mechanical and chemical cues such that it can signal in a sensitive, responsive and robust manner. In this Review, we discuss how SAC proteins interact to allow production of the mitotic checkpoint complex (MCC) that halts anaphase progression by inhibiting the anaphase-promoting complex/cyclosome (APC/C). We highlight recent advances aimed at understanding the dynamic signalling properties of the SAC and how it interprets various naturally occurring intermediate attachment states. Further, we discuss SAC signalling in the context of the mammalian multisite kinetochore and address the impact of the fibrous corona. We also identify current challenges in understanding how the SAC ensures high-fidelity chromosome segregation.

Association of Hsp90 with p53 and Fizzy related homolog (Fzr) synchronizing Anaphase Promoting Complex (APC/C): An unexplored ally towards oncogenic pathway.

The intricate molecular interactions leading to the oncogenic pathway are the consequence of cell cycle modification controlled by a bunch of cell cycle regulatory proteins. The tumor suppressor and cell cycle regulatory proteins work in coordination to maintain a healthy cellular environment. The integrity of this cellular protein pool is perpetuated by heat shock proteins/chaperones, which assist in proper protein folding during normal and cellular stress conditions. Among these versatile groups of chaperone proteins, Hsp90 is one of the significant ATP-dependent chaperones that aid in stabilizing many tumor suppressors and cell cycle regulator protein targets. Recently, studies have revealed that in cancerous cell lines, Hsp90 stabilizes mutant p53, 'the guardian of the genome.' Hsp90 also has a significant impact on Fzr, an essential regulator of the cell cycle having an important role in the developmental process of various organisms, including Drosophila, yeast, Caenorhabditis elegans, and plants. During cell cycle progression, p53 and Fzr coordinately regulate the Anaphase Promoting Complex (APC/C) from metaphase to anaphase transition up to cell cycle exit. APC/C mediates proper centrosome function in the dividing cell. The centrosome acts as the microtubule organizing center for the correct segregation of the sister chromatids to ensure perfect cell division. This review examines the structure of Hsp90 and its co-chaperones, which work in synergy to stabilize proteins such as p53 and Fizzy-related homolog (Fzr) to synchronize the Anaphase Promoting Complex (APC/C). Dysfunction of this process activates the oncogenic pathway leading to the development of cancer. Additionally, an overview of current drugs targeting Hsp90 at various phases of clinical trials has been included.

APC/CCDC20 targets SCFFBL17 to activate replication stress responses in Arabidopsis.

DNA replication stress threatens genome stability and affects plant growth and development. How plants resolve replication stress is poorly understood. The protein kinase WEE1-mediated cell cycle arrest is required for replication stress responses. The E3 ubiquitin ligases anaphase-promoting complex/cyclosome (APC/C) and Skp1/Cullin 1/F-box (SCF) are essential regulators of the cell cycle. Here, we show that APC/CCDC20 mediates the degradation of SCFFBL17 during replication stress responses in Arabidopsis thaliana. Biochemically, WEE1 interacts with and phosphorylates the APC/C co-activator APC10, which enhances the interaction between F-BOX-LIKE17 (FBL17) and CELL DIVISION CYCLE 20 (CDC20), an activator of APC/C. Both APC10 and CDC20 are required for the polyubiquitination and degradation of FBL17. Genetically, silencing CDC20 or APC10 confers plant hypersensitivity to replication stress, which is suppressed by loss of FBL17. Collectively, our study suggests that WEE1 activates APC/C to inhibit FBL17, providing insight into replication stress responses in plants.

Plant lineage-specific PIKMIN1 drives APC/CCCS52A2 E3-ligase activity-dependent cell division.

The anaphase-promoting complex/cyclosome (APC/C) marks key cell cycle proteins for proteasomal breakdown, thereby ensuring unidirectional progression through the cell cycle. Its target recognition is temporally regulated by activating subunits, one of which is called CELL CYCLE SWITCH 52 A2 (CCS52A2). We sought to expand the knowledge on the APC/C by using the severe growth phenotypes of CCS52A2-deficient Arabidopsis (Arabidopsis thaliana) plants as a readout in a suppressor mutagenesis screen, resulting in the identification of the previously undescribed gene called PIKMIN1 (PKN1). PKN1 deficiency rescues the disorganized root stem cell phenotype of the ccs52a2-1 mutant, whereas an excess of PKN1 inhibits the growth of ccs52a2-1 plants, indicating the need for control of PKN1 abundance for proper development. Accordingly, the lack of PKN1 in a wild-type background negatively impacts cell division, while its systemic overexpression promotes proliferation. PKN1 shows a cell cycle phase-dependent accumulation pattern, localizing to microtubular structures, including the preprophase band, the mitotic spindle, and the phragmoplast. PKN1 is conserved throughout the plant kingdom, with its function in cell division being evolutionarily conserved in the liverwort Marchantia polymorpha. Our data thus demonstrate that PKN1 represents a novel, plant-specific protein with a role in cell division that is likely proteolytically controlled by the CCS52A2-activated APC/C.

Septin 9 controls CCNB1 stabilization via APC/CCDC20 during meiotic metaphase I/anaphase I transition in mouse oocytes.

The anaphase promoting complex/cyclosome (APC/C) and its cofactors CDH1 and CDC20 regulate the accumulation/degradation of CCNB1 during mouse oocyte meiotic maturation. Generally, the CCNB1 degradation mediated by APC/CCDC20 activity is essential for the transition from metaphase to anaphase. Here, by using siRNA and mRNA microinjection, as well as time-lapse live imaging, we showed that Septin 9, which mediates the binding of septins to microtubules, is critical for oocyte meiotic cell cycle progression. The oocytes were arrested at the MI stage and the connection between chromosome kinetochores and spindle microtubules was disrupted after Septin 9 depletion. As it is well known that spindle assembly checkpoint (SAC) is an important regulator of the MI-AI transition, we thus detected the SAC activity and the expression of CDC20 and CCNB1 which were the downstream proteins of SAC during this critical period. The signals of Mad1 and BubR1 still remained on the kinetochores of chromosomes in Septin 9 siRNA oocytes at 9.5 h of in vitro culture when most control oocytes entered anaphase I. The expression of CCNB1 did not decrease and the expression of CDC20 did not increase at 9.5 h in Septin 9 siRNA oocytes. Microinjection of mRNA encoding Septin 9 or CDC20 could partially rescue MI arrest caused by Septin 9 siRNA. These results suggest that Septin 9 is required for meiotic MI-AI transition by regulating the kinetochore-microtubule connection and SAC protein localization on kinetochores, whose effects are transmitted to APC/CCDC20 activity and CCNB1 degradation in mouse oocytes.

APC/CCdc20-mediated degradation of Clb4 prompts astral microtubule stabilization at anaphase onset.

Key for accurate chromosome partitioning to the offspring is the ability of mitotic spindle microtubules to respond to different molecular signals and remodel their dynamics accordingly. Spindle microtubules are conventionally divided into three classes: kinetochore, interpolar, and astral microtubules (kMTs, iMTs, and aMTs, respectively). Among all, aMT regulation remains elusive. Here, we show that aMT dynamics are tightly regulated. aMTs remain unstable up to metaphase and are stabilized at anaphase onset. This switch in aMT dynamics, important for proper spindle orientation, specifically requires the degradation of the mitotic cyclin Clb4 by the Anaphase Promoting Complex bound to its activator subunit Cdc20 (APC/CCdc20). These data highlight a unique role for mitotic cyclin Clb4 in controlling aMT regulating factors, of which Kip2 is a prime candidate, provide a framework to understand aMT regulation in vertebrates, and uncover mechanistic principles of how the APC/CCdc20 choreographs the timing of late mitotic events by sequentially impacting on the three classes of spindle microtubules.

The anaphase promoting complex/cyclosome ubiquitylates histone H2B on the promoter during UbcH10 transactivation.

Among various post-translational modifications of histones, ubiquitylation plays a crucial role in transcription regulation. Histone mono-ubiquitylation by RING finger motif-containing ubiquitin ligases is documented in this respect. The RING finger ligases primarily regulate the cell cycle, where the anaphase-promoting complex/cyclosome (APC/C) takes charge as mitotic ubiquitin machinery. Reportedly, APC/C participates in transcriptional activation of the ubiquitin carrier protein UbcH10. However, the ubiquitylation activity of APC/C on the UBCH10 promoter remains elusive. This study shows that APC/C, with its adapter protein Cdc20, catalyses mono-ubiquitylation of Lysine-120 in histone 2B on the UBCH10 promoter. This study also identified a cell-cycle-specific pattern of this modification. Finally, APC/C-driven crosstalk of acetylation and ubiquitylation was found operational on UBCH10 trans-regulation. Together, these findings suggest a role for APC/C catalysed promoter ubiquitylation in managing transcription of cell cycle regulatory genes.

Current Progress and Perspectives of CDC20 in Female Reproductive Cancers.

The cancers of the cervix, endometrium, ovary, and breast are great threats to women's health. Cancer is characterized by the uncontrolled proliferation of cells and deregulated cell cycle progression is one of the main causes of malignancy. Agents targeting cell cycle regulators may have potential anti-tumor effects. CDC20 (cell division cycle 20 homologue) is a co-activator of the anaphase-promoting complex/cyclosome (APC/C) and thus acts as a mitotic regulator. In addition, CDC20 serves as a subunit of the mitotic checkpoint complex (MCC) whose function is to inhibit APC/C. Recently, higher expression of CDC20 has been reported in these cancers and was closely associated with their clinicopathological parameters, indicating CDC20 a potential target for cancer treatment that is worth further study. In the present review, we summarized current progress and put forward perspectives of CDC20 in female reproductive cancers.