Anaphase-promoting Complex Research Articles

APC/C Ubiquitin Ligase: Coupling Cellular Differentiation to G1/G0 Phase in Multicellular Systems.

The anaphase-promoting complex/cyclosome (APC/C) is an evolutionarily conserved ubiquitin ligase that controls cell cycle progression through spatiotemporally regulated proteolysis. Although recent studies revealed its postmitotic function, our knowledge of the role of APC/C beyond cell cycle regulation in the biology of multicellular organisms is far from complete. Here, I review recent advances in the function of APC/C in animal development, specifically focusing on its emerging role in regulating cell differentiation. I describe how APC/C regulates distinct processes during the course of differentiation by deploying diverse molecular machineries in a variety of developmental contexts. Also, I discuss the significance and clinical relevance of the unique capacity of APC/C and other cell cycle regulators to couple distinct cellular processes with cell proliferation control.

Identification of the potential therapeutic target gene UBE2C in human hepatocellular carcinoma: An investigation based on GEO and TCGA databases.

Hepatocellular carcinoma (HCC) ranks the third major cause of cancer-associated mortality globally. Numerous studies have attempted to elucidate the underlying mechanisms of HCC using various biomarkers. In the present study, two expression profiles datasets from Gene Expression Omnibus (GSE76427 and GSE84402) and data associated with liver cancer samples from The Cancer Genome Atlas (TCGA) were downloaded for integrated analysis. Five differentially expressed genes (DEGs) exhibiting high expression, including ubiquitin-conjugating enzyme 2C (UBE2C), topoisomerase II α, pituitary tumor transforming gene 1, glypican-3 and polycomb-repressive complex 1, were selected and considered as candidate genes. Enrichment analysis demonstrated that these genes were associated with Gene Ontology terms of cellular components and molecular functions, including regulation of apoptosis, stabilization of p53 and Anaphase Promoting Complex/Cyclosome (APC/C) (APC/C:Cdc20)-mediated degradation of Securin. The expression profiles of these genes in HCC, other human malignancies and different human HCC cell lines were validated using GSE14520, GSE3500 and TCGA data. The results confirmed the upregulation of UBE2C in tissues from patients with HCC or other human malignancies and human liver cancer cell lines, compared with the expression levels in the corresponding adjacent non-tumor tissues and cell lines, respectively. Patients with HCC who exhibited an increased messenger RNA level of UBE2C exhibited a significantly shorter survival time. The results of the present study suggest that the overexpression of UBE2C may be used as a novel prognostic biomarker of HCC.

Characterization of Cks2 localization and interaction with Cdk1 in mitotic cells of Xenopus laevis embryos

Although mitotic regulation has been extensively studied, several of the mechanisms behind this process have yet to be fully understood. Our group is particularly interested in mechanisms regulating the timing of mitotic exit and how such mechanisms coordinate with other mitotic cues such as spindle orientation. Recent work in our lab has shown that spindle movements in epithelial cells of the developing frog embryo correlate with mitotic exit and perturbations in normal spindle movements lengthen metaphase. Further, these changes in mitotic progression are accompanied by increases in Cdk1 Tyr15 phosphorylation, specifically at cell junctions. We propose that the link between spindle movement and mitotic progression is an important part of the mechanisms influencing spindle orientation in the frog epithelium. Our current work examines the role of Cks2 in the aforementioned mechanism. Cks2 serves as a targeting subunit for the mitotic kinase Cdk1. Specifically, work in yeast suggests Cks2 may direct Cdk1 toward the anaphase‐promoting complex (APC) and interact with both complexes, facilitating the phosphorylation of Cdk1 and activation of the APC and thus help control mitotic exit. In this study, we characterize Cks2's mitotic function in Xenopus laevis. Our aim is to characterize Cks2 distribution throughout mitosis and determine whether perturbing Cks2 function alters mitotic progression in the gastrula‐stage epithelium. We hypothesize that if Cks2 influences Cdk1 function in mitosis, then the two proteins will exhibit similar localization patterns in mitotic cells. We visualized endogenous Cks2 and also expressed GFP‐tagged Cks2 in fixed epithelial tissues of Xenopus laevis embryos through confocal microscopy. Our early data show that Cks2 localization overlaps with that of a phosphorylated Cdk1(pY15) at cell junctions and overexpression of GFP‐tagged Cks2 results in increased levels of Cdk1‐pY15. In addition, the assay expressing a dominant negative version of Cks2 suggests that depletion of endogenous Cks2 activity may contribute to decreased Cdk1‐pY15 at cell junctions and increased cell area. Therefore, these data suggest a link between Cks2 and Cdk1 in mitotic regulation and are a first step toward a greater characterization of Cks2's role in regulating mitotic progression in vertebrate organisms.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Elevated signature of a gene module coexpressed with CDC20 marks genomic instability in glioma

Genomic instability (GI) drives tumor heterogeneity and promotes tumor progression and therapy resistance. However, causative factors underlying GI and means for clinical detection of GI in glioma are inadequately identified. We describe here that elevated expression of a gene module coexpressed with CDC20 (CDC20-M), the activator of the anaphase-promoting complex in the cell cycle, marks GI in glioma. The CDC20-M, containing 139 members involved in cell proliferation, DNA damage response, and chromosome segregation, was found to be consistently coexpressed in glioma transcriptomes. The coexpression of these genes was conserved across multiple species and organ systems, particularly in human neural stem and progenitor cells. CDC20-M expression was not correlated with the morphological subtypes, nor with the recently defined molecular subtypes of glioma. CDC20-M signature was an independent and robust predictor for poorer prognosis in over 1,000 patients from four large databases. Elevated CDC20-M signature enabled the identification of individual glioma samples with severe chromosome instability and mutation burden and of primary glioma cell lines with extensive mitotic errors leading to chromosome mis-segregation. AURKA, a core member of CDC20-M, was amplified in one-third of CDC20-M-high gliomas with gene-dosage-dependent expression. MLN8237, a Food and Drug Administration-approved AURKA inhibitor, selectively killed temozolomide-resistant primary glioma cells in vitro and prolonged the survival of a patient-derived xenograft mouse model with a high-CDC20-M signature. Our findings suggest that application of the CDC20-M signature may permit more selective use of adjuvant therapies for glioma patients and that dysregulated CDC20-M members may provide a therapeutic vulnerability in glioma.

Blocking Mitotic Exit of Ovarian Cancer Cells by Pharmaceutical Inhibition of the Anaphase-Promoting Complex Reduces Chromosomal Instability

Paclitaxel is a frontline drug for the treatment of epithelial ovarian cancer (EOC). However, following paclitaxel-platinum based chemotherapy, tumor recurrence occurs in most ovarian cancer patients. Chromosomal instability (CIN) is a hallmark of cancer and represents genetic variation fueling tumor adaptation to cytotoxic effects of anticancer drugs. In this study, our Kaplan-Meier analysis including 263 ovarian cancer patients (stages I/II) revealed that high Polo-like kinase (PLK) 1 expression correlates with bad prognosis. To evaluate the role of PLK1 as potential cancer target within a combinatorial trial, we induced strong mitotic arrest in ovarian cancer cell lines by synergistically co-targeting microtubules (paclitaxel) and PLK1 (BI6727) followed by pharmaceutical inhibition of the Anaphase-Promoting Complex (APC/C) using proTAME. In short- and long-term experiments, this triple treatment strongly activated apoptosis in cell lines and primary ovarian cells derived from cancer patients. Mechanistically, BI6727/paclitaxel/proTAME stabilize Cyclin B1 and trigger mitotic arrest, which initiates mitochondrial apoptosis by inactivation of antiapoptotic BCL-2 family proteins, followed by activation of caspase-dependent effector pathways. This triple treatment prevented endoreduplication and reduced CIN, two mechanisms that are associated with aggressive tumors and the acquisition of drug resistance. This “two-punch strategy” (strong mitotic arrest followed by blocking mitotic exit) has important implications for developing paclitaxel-based combinatorial treatments in ovarian cancer.

Short-term starvation stress at young adult stages enhances meiotic activity of germ cells to maintain spermatogenesis in aged male Caenorhabditis elegans.

To survive and reproduce, living organisms must evolve numerous mechanisms to re‐adjust their physiology when encountering adverse conditions that subject them to severe stress. We found that short‐term starvation (STS) stress in young adult male Caenorhabditis elegans can significantly improve their vitality (relative to nonstressed males) when they are aged. In addition, we found that stress‐treated aged males maintained reproductive activity equivalent to young males, whereas nonstressed aged males quickly lost reproductive ability. STS stress can preserve sperm number and quality in aged male worms. Spermatogenesis involves germ cell mitosis and meiosis. We found that germ cell meiotic activity is more sensitive to aging than mitotic activity and is declining rapidly with age. We examined the role of numerous factors important for spermatogenesis on STS‐preserved spermatogenesis during aging. Our results show that mutant strains deficient in anaphase‐promoting complex/cyclosome (APC/C) function fail to exhibit the STS stress‐enhanced spermatogenesis found in wild‐type N2 worms, suggesting that the mechanism underlying starvation‐induced spermatogenesis involves the APC/C complex, a conserved ubiquitin‐protein ligase E3 complex. Furthermore, transgenic expression of FZY‐1/CDC‐20, a coactivator of APC/C, ameliorated the age‐associated decline of meiosis, similar to the hormetic effect of STS.

Candidate genes and potential mechanisms for chemoradiotherapy sensitivity in locally advanced rectal cancer.

The aim of the present study was to investigate candidate genes for chemoradiotherapy (CRT) sensitivity in patients with locally advanced rectal cancer (LARC), and the potential mechanisms of their action. A microarray dataset (GSE98959) was obtained from the Gene Expression Omnibus database that included microRNA (miRNA, miR) expression profiling of 22 samples from patients with LARC who had received preoperative radiotherapy and chemotherapy. Of these patients, 10 responded to the treatment and 12 did not. Differentially expressed miRNAs (DEMs) were identified, followed by the construction of an miRNA-gene network. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) function analyses were performed on the target genes in the miRNA-gene network. Furthermore, a protein-protein interaction (PPI) network was constructed on the basis of the target genes, followed by GO function enrichment and KEGG pathway analysis. A total of 30 DEMs were identified between the responder and non-responder groups. Thiamine metabolism (including miR-371a-3p) was the pathway with the highest enrichment of DEMs. The pathway that was most markedly enriched in the target genes of upregulated miRNAs was the pluripotency of stem cells pathway, as indicated by phosphoinositide-4,5-bisphosphate 3-kinase γ (PIK3CG) and anaphase-promoting complex subunit 2 (APC2). Pathways in cancer exhibited the highest enrichment in the set of target genes of downregulated miRNAs. KEGG pathway and GO function analysis indicated that target genes in the PPI network were enriched in the glioma pathway and assembled in the intracellular signaling cascade function, as indicated by the proto-oncogene NRAS. miR-371a-3p may be a candidate miRNA for CRT sensitivity in LARC via the thiamine metabolism pathway. PIK3CG and APC2 may contribute to CRT sensitivity via signaling pathways regulating the pluripotency of stem cells. Furthermore, NRAS may serve an important role in mediating CRT sensitivity via an intracellular signaling cascade.

Cyclin B3 is required for metaphase to anaphase transition in oocyte meiosis I.

Meiosis with a single round of DNA replication and two successive rounds of chromosome segregation requires specific cyclins associated with cyclin-dependent kinases (CDKs) to ensure its fidelity. But how cyclins control the distinctive meiosis is still largely unknown. In this study, we explored the role of cyclin B3 in female meiosis by generating Ccnb3 mutant mice via CRISPR/Cas9. Ccnb3 mutant oocytes characteristically arrested at metaphase I (MetI) with normal spindle assembly and lacked enough anaphase-promoting complex/cyclosome (APC/C) activity, which is spindle assembly checkpoint (SAC) independent, to initiate anaphase I (AnaI). Securin siRNA or CDK1 inhibitor supplements rescued the MetI arrest. Furthermore, CCNB3 directly interacts with CDK1 to exert kinase function. Besides, the MetI arrest oocytes had normal development after intracytoplasmic sperm injection (ICSI) or parthenogenetic activation (PA), along with releasing the sister chromatids, which implies that Ccnb3 exclusively functioned in meiosis I, rather than meiosis II. Our study sheds light on the specific cell cycle control of cyclins in meiosis.

Cell-Size-Independent Spindle Checkpoint Failure Underlies Chromosome Segregation Error in Mouse Embryos

Chromosome segregation errors during mammalian preimplantation development cause "mosaic" embryos comprising a mixture of euploid and aneuploid cells, which reduce the potential for a successful pregnancy [1-5], but why these errors are common is unknown. In most cells, chromosome segregation error is averted by the spindle assembly checkpoint (SAC), which prevents anaphase-promoting complex (APC/C) activation and anaphase onset until chromosomes arealigned with kinetochores attached to spindle microtubules [6, 7], but little is known about the SAC's role in the early mammalian embryo. In C.elegans, the SAC is weak in early embryos, and it strengthens during early embryogenesis as a result of progressively lessening cell size [8, 9]. Here, using live imaging, micromanipulation, gene knockdown, and pharmacological approaches, we show that this is not the case in mammalian embryos. Misaligned chromosomes in the early mouse embryo can recruit SAC components to mount a checkpoint signal, but this signal fails to prevent anaphase onset, leading to high levels of chromosome segregation error. We find that failure of the SAC to prolong mitosis is not attributable to cell size. We show that mild chemical inhibition of APC/C can extend mitosis, thereby allowing more time for correct chromosome alignment and reducing segregation errors. SAC-APC/C disconnect thus presents a mechanistic explanation for frequent chromosome segregation errors in early mammalian embryos. Moreover, our data provide proof of principle that modulation of the SAC-APC/C axis can increase the likelihood of error-free chromosome segregation in cultured mammalian embryos.

Anaphase-promoting complex/cyclosome regulates RdDM activity by degrading DMS3 in Arabidopsis

During RNA-directed DNA methylation (RdDM), the DDR complex, composed of DRD1, DMS3, and RDM1, is responsible for recruiting DNA polymerase V (Pol V) to silence transposable elements (TEs) in plants. However, how the DDR complex is regulated remains unexplored. Here, we show that the anaphase-promoting complex/cyclosome (APC/C) regulates the assembly of the DDR complex by targeting DMS3 for degradation. We found that a substantial set of RdDM loci was commonly de-repressed in apc/c and pol v mutants, and that the defects in RdDM activity resulted from up-regulated DMS3 protein levels, which finally caused reduced Pol V recruitment. DMS3 was ubiquitinated by APC/C for degradation in a D box-dependent manner. Competitive binding assays and gel filtration analyses showed that a proper level of DMS3 is critical for the assembly of the DDR complex. Consistent with the importance of the level of DMS3, overaccumulation of DMS3 caused defective RdDM activity, phenocopying the apc/c and dms3 mutants. Moreover, DMS3 is expressed in a cell cycle-dependent manner. Collectively, these findings provide direct evidence as to how the assembly of the DDR complex is regulated and uncover a safeguarding role of APC/C in the regulation of RdDM activity.

Cdh1-Mediated Metabolic Switch from Pentose Phosphate Pathway to Glycolysis Contributes to Sevoflurane-Induced Neuronal Apoptosis in Developing Brain.

Cdh1 is a regulatory subunit of the anaphase promoting complex/cyclosome (APC/C), known to be involved in regulating neuronal survival. The role of Cdh1 in volatile anesthetics-induced neuronal apoptosis in the developing brain is unknown. In this study, we used postnatal day 7 (P7) and day 21 (P21) mice exposed to 2.3% sevoflurane for 6 h to investigate at which age and duration of exposure sevoflurane affects the expression of Cdh1 and glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) and that of the pentose phosphate pathway (PPP) enzyme, glucose-6-phosphate dehydrogenase (G6PD). Furthermore, we tested whether the cyclin-dependent kinases (cdks) inhibitor roscovatine could counteract the effects caused by exposure to sevoflurane. Finally, we applied the glycolysis inhibitor 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3-PO), G6PD inhibitor dehydroepiandrosterone (DHEA), and exogenous reduced glutathione to examine the contribution of the glycolysis pathway and PPP to sevoflurane-induced neuroapoptosis. We found that prolonged sevoflurane anesthesia significantly reduces the Cdh1 level in P7 mice compared to in the P21 ones; moreover, the decrease in Cdh1 level results in a switch in glucose metabolism from the PPP to neuronal glycolysis. This leads to an imbalance between reactive oxygen species production and reduced glutathione level in the developing brain, which is more susceptible to oxidative stress. As a result, sevoflurane induces neuroapoptosis through Cdh1-mediated glucose metabolism reprogramming. Our study demonstrates a critical role of Cdh1 in sevoflurane-induced neuroapoptosis by shifting PPP to the glycolytic pathway in the developing brain. These findings suggest that Cdh1 may be a novel target for preventing volatile anesthetics-induced neurotoxicity and memory impairment.

The anaphase-promoting complex regulates the degradation of the inner nuclear membrane protein Mps3.

The nucleus is enclosed by the inner nuclear membrane (INM) and the outer nuclear membrane (ONM). While the ONM is continuous with the endoplasmic reticulum (ER), the INM is independent and separates the nucleoplasm from the ER lumen. Turnover of ER proteins has been well characterized by the ER-associated protein degradation (ERAD) pathway, but very little is known about turnover of resident INM proteins. Here we show that the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase, regulates the degradation of Mps3, a conserved integral protein of the INM. Turnover of Mps3 requires the ubiquitin-conjugating enzyme Ubc7, but was independent of the known ERAD ubiquitin ligases Doa10 and Hrd1 as well as the recently discovered Asi1-Asi3 complex. Using a genetic approach, we have found that Cdh1, a coactivator of APC/C, modulates Mps3 stability. APC/C controls Mps3 degradation through Mps3's N terminus, which resides in the nucleoplasm and possesses two putative APC/C-dependent destruction motifs. Accumulation of Mps3 at the INM impairs nuclear morphological changes and cell division. Our findings therefore reveal an unexpected mechanism of APC/C-mediated protein degradation at the INM that coordinates nuclear morphogenesis and cell cycle progression.

Cyclin B3 promotes anaphase I onset in oocyte meiosis.

Meiosis poses unique challenges because two rounds of chromosome segregation must be executed without intervening DNA replication. Mammalian cells express numerous temporally regulated cyclins, but how these proteins collaborate to control meiosis remains poorly understood. Here, we show that female mice genetically ablated for cyclin B3 are viable-indicating that the protein is dispensable for mitotic divisions-but are sterile. Mutant oocytes appear normal until metaphase I but then display a highly penetrant failure to transition to anaphase I. They arrest with hallmarks of defective anaphase-promoting complex/cyclosome (APC/C) activation, including no separase activity, high CDK1 activity, and high cyclin B1 and securin levels. Partial APC/C activation occurs, however, as exogenously expressed APC/C substrates can be degraded. Cyclin B3 forms active kinase complexes with CDK1, and meiotic progression requires cyclin B3-associated kinase activity. Cyclin B3 homologues from frog, zebrafish, and fruit fly rescue meiotic progression in cyclin B3-deficient mouse oocytes, indicating conservation of the biochemical properties and possibly cellular functions of this germline-critical cyclin.

Concerted expression of a cell cycle regulator and a metabolic enzyme from a bicistronic transcript in plants

Eukaryotic mRNAs frequently contain upstream open reading frames (uORFs), encoding small peptides that may control translation of the main ORF (mORF). Here, we report the characterization of a distinct bicistronic transcript in Arabidopsis. We analysed loss-of-function phenotypes of the inorganic polyphosphatase TRIPHOSPHATE TUNNEL METALLOENZYME 3 (AtTTM3), and found that catalytically inactive versions of the enzyme could fully complement embryo and growth-related phenotypes. We could rationalize these puzzling findings by characterizing a uORF in the AtTTM3 locus encoding CELL DIVISION CYCLE PROTEIN 26 (CDC26), an orthologue of the cell cycle regulator. We demonstrate that AtCDC26 is part of the plant anaphase promoting complex/cyclosome (APC/C), regulates accumulation of APC/C target proteins and controls cell division, growth and embryo development. AtCDC26 and AtTTM3 are translated from a single transcript conserved across the plant lineage. While there is no apparent biochemical connection between the two gene products, AtTTM3 coordinates AtCDC26 translation by recruiting the transcript into polysomes. Our work highlights that uORFs may encode functional proteins in plant genomes.

La sous-unité catalytique de l’anaphase-promoting complex APC11 est impliquée dans la progression tumorale des cancers colorectaux présentant une instabilité chromosomique

La sous-unité catalytique de l’<b><i>anaphase-promoting complex</i></b> APC11 est impliquée dans la progression tumorale des cancers colorectaux présentant une instabilité chromosomique

Regulation of OLC1 protein expression by the anaphase-promoting complex.

Overexpressed in lung cancer 1 (OLC1) is a potential oncogene overexpressed in human lung cancer and in other types of malignant tumor. The elevated expression of OLC1 contributes to tumor genesis and progression. However, the mechanisms regulating the expression of OLC1 remain unclear. In the present study, using lung and esophageal cancer cell lines, it was demonstrated that OLC1 was a short-lived, cell cycle-dependent protein regulated through the anaphase-promoting complex/cyclosome (APC/c)-ubiquitin pathway by directly interacting with the APC2 subunit. Through the action of two co activator proteins, cadherin 1 (Cdh1) and cell-division cycle protein 20 (Cdc20), the OLC1 protein was ubiquitinated and degraded. Following treatment with a proteasome inhibitor, OLC1 protein levels were elevated. Inversely, the upregulation of Cdh1 and Cdc20 facilitated OLC1 degradation. By inducing point mutations of the assumed degradation motif of OLC1, it was revealed that an intact destruction (D)-box was necessary. As expected, the D-box-mutated OLC1 exhibited a higher capacity for promoting cell growth and clone formation. Collectively, these findings indicate that the expression of the candidate oncogene OLC1 is cell cycle-dependent and is regulated by an APC/c mediated ubiquitin-proteasome pathway.

The Anaphase Promoting Complex/Cyclosome (APC/C): A Versatile E3 Ubiquitin Ligase.

In the present chapter we discuss the essential roles of the human E3 ubiquitin ligase Anaphase Promoting Complex/Cyclosome (APC/C) in mitosis as well as the emerging evidence of important APC/C roles in cellular processes beyond cell division control such as regulation of genomic integrity and cell differentiation of the nervous system. We consider the potential incipient role of APC/C dysregulation in the pathophysiology of the neurological disorder Alzheimer's disease (AD). We also discuss how certain Deoxyribonucleic Acid (DNA) and Ribonucleic Acid (RNA) viruses take control of the host's cell division regulatory system through harnessing APC/C ubiquitin ligase activity and hypothesise the plausible molecular mechanisms underpinning virus manipulation of the APC/C. We also examine how defects in the function of this multisubunit protein assembly drive abnormal cell proliferation and lastly argue the potential of APC/C as a promising therapeutic target for the development of innovative therapies for the treatment of chronic malignancies such as cancer.

Single-molecule methods for measuring ubiquitination and protein stability.

The ubiquitin-proteasome system (UPS) contributes to changes in cell state and homeostatic maintenance in humans by modulating the stability of about a third of human proteins. For example, cell-cycle regulation requires a central ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C), which starts a ubiquitination cascade leading to the degradation of multiple targets. This targeted degradation is mediated by the 26S proteasome, a 2.5-MDa protein complex, which recognizes and degrades ubiquitinated proteins at rates partially controlled by the variations in ubiquitin chain topology. Substrate selectivity of ubiquitin ligases such as the APC/C and of the 26S proteasome from pools of near-identical targets reflects highly regulated kinetic mechanisms. Single-molecule techniques are powerful tools that allow distinction between differential substrate affinities and identification of reaction intermediates in complex mixtures. Here we describe fluorescence-based single-molecule imaging of in vitro ubiquitination reactions catalyzed by the APC/C and ubiquitin-dependent degradation reactions catalyzed by the 26S proteasome.

The Phosphatase PP1 Promotes Mitotic Slippage Through Mad3 Dephosphorylation

Accurate chromosome segregation in mitosis requires the bipolar attachment of kinetochores to spindle microtubules. A conserved surveillance mechanism, the Spindle Assembly Checkpoint (SAC), responds to lack of kinetochore-microtubule connections and delays the onset of anaphase until all chromosomes are bipolarly attached. SAC signalling fires at kinetochores and involves the formation of a soluble Mitotic Checkpoint Complex (MCC) made by Mad2, Mad3/BubR1, Bub3 and Cdc20, which inhibits the ubiquitin ligase activity of the Anaphase Promoting Complex (APC). The mitotic delay imposed by SAC, however, is not everlasting. After a variable time during which kinetochores have failed to establish bipolar connections, cells escape from the SAC-induced mitotic arrest through a process called mitotic slippage. Mitotic slippage occurs in the presence of SAC signalling at kinetochores, but whether and how MCC stability and APC inhibition are actively controlled during slippage is not known. The PP1 phosphatase has emerged as a key factor in SAC silencing once all kinetochores are bipolarly attached. PP1 turns off SAC signalling through dephosphorylation of the SAC scaffold Knl1/Blinkin at kinetochores. Here we show that in budding yeast PP1 is also required for mitotic slippage. However, its involvement in this process is not linked to kinetochores but rather to MCC stability. We identify S268 of Mad3 as a critical target of PP1 in this process. Mad3 S268 dephosphorylation destabilises the MCC, but in spite of that, does not affect the initial SAC-induced mitotic arrest. Conversely, it accelerates mitotic slippage and overcomes the slippage defect of PP1 mutants. Thus, slippage is not the mere consequence of incomplete APC inactivation that brings about mitotic exit, as originally proposed, but involves the exertive antagonism between kinases and phosphatases.

Genetic analysis reveals functions of atypical polyubiquitin chains

Although polyubiquitin chains linked through all lysines of ubiquitin exist, specific functions are well-established only for lysine-48 and lysine-63 linkages in Saccharomyces cerevisiae. To uncover pathways regulated by distinct linkages, genetic interactions between a gene deletion library and a panel of lysine-to-arginine ubiquitin mutants were systematically identified. The K11R mutant had strong genetic interactions with threonine biosynthetic genes. Consistently, we found that K11R mutants import threonine poorly. The K11R mutant also exhibited a strong genetic interaction with a subunit of the anaphase-promoting complex (APC), suggesting a role in cell cycle regulation. K11-linkages are important for vertebrate APC function, but this was not previously described in yeast. We show that the yeast APC also modifies substrates with K11-linkages in vitro, and that those chains contribute to normal APC-substrate turnover in vivo. This study reveals comprehensive genetic interactomes of polyubiquitin chains and characterizes the role of K11-chains in two biological pathways.