RESUMO
Regulated cell cycle progression ensures homeostasis and prevents cancer. In proliferating cells, premature S phase entry is avoided by the E3 ubiquitin ligase anaphasepromoting complex/cyclosome (APC/C), although the APC/C substrates whose degradation restrains G1-S progression are not fully known. The APC/C is also active in arrested cells that exited the cell cycle, but it is not clear whether APC/C maintains all types of arrest. Here, by expressing the APC/C inhibitor, EMI1, we show that APC/C activity is essential to prevent S phase entry in cells arrested by pharmacological cyclin-dependent kinases 4 and 6 (CDK4/6) inhibition (Palbociclib). Thus, active protein degradation is required for arrest alongside repressed cell cycle gene expression. The mechanism of rapid and robust arrest bypass from inhibiting APC/C involves CDKs acting in an atypical order to inactivate retinoblastoma-mediated E2F repression. Inactivating APC/C first causes mitotic cyclin B accumulation which then promotes cyclin A expression. We propose that cyclin A is the key substrate for maintaining arrest because APC/C-resistant cyclin A, but not cyclin B, is sufficient to induce S phase entry. Cells bypassing arrest from CDK4/6 inhibition initiate DNA replication with severely reduced origin licensing. The simultaneous accumulation of S phase licensing inhibitors, such as cyclin A and geminin, with G1 licensing activators disrupts the normal order of G1-S progression. As a result, DNA synthesis and cell proliferation are profoundly impaired. Our findings predict that cancers with elevated EMI1 expression will tend to escape CDK4/6 inhibition into a premature, underlicensed S phase and suffer enhanced genome instability.
Assuntos
Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Humanos , Quinase 6 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/genética , Linhagem Celular Tumoral , Fase S/efeitos dos fármacos , Piridinas/farmacologia , Piperazinas/farmacologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Fatores de Transcrição E2F/metabolismo , Fatores de Transcrição E2F/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Ciclinas/metabolismo , Ciclinas/genética , Proteínas F-BoxRESUMO
O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is the sole enzyme that catalyzes all O-GlcNAcylation reactions intracellularly. Previous investigations have found that OGT levels oscillate during the cell division process. Specifically, OGT abundance is downregulated during mitosis, but the underlying mechanism is lacking. Here we demonstrate that OGT is ubiquitinated by the ubiquitin E3 ligase, anaphase promoting complex/cyclosome (APC/C)-cell division cycle 20 (Cdc20). We show that APC/CCdc20 interacts with OGT through a conserved destruction box (D-box): Arg-351/Leu-354, the abrogation of which stabilizes OGT. As APC/CCdc20-substrate binding is often preceded by a priming ubiquitination event, we also used mass spectrometry and mapped OGT Lys-352 to be a ubiquitination site, which is a prerequisite for OGT association with APC/C subunits. Interestingly, in The Cancer Genome Atlas, R351C is a uterine carcinoma mutant, suggesting that mutations of the D-box are linked with tumorigenesis. Paradoxically, we found that both R351C and the D-box mutants (R351A/L354A) inhibit uterine carcinoma in mouse xenograft models, probably due to impaired cell division and proliferation. In sum, we propose a model where OGT Lys-352 ubiquitination primes its binding with APC/C, and then APC/CCdc20 partners with OGT through the D-box for its mitotic destruction. Our work not only highlights the key mechanism that regulates OGT during the cell cycle, but also reveals the mutual coordination between glycosylation and the cell division machinery.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase , Mitose , N-Acetilglucosaminiltransferases , Ubiquitinação , Humanos , N-Acetilglucosaminiltransferases/metabolismo , N-Acetilglucosaminiltransferases/genética , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/genética , Animais , Proteínas Cdc20/metabolismo , Proteínas Cdc20/genética , Camundongos , Proteólise , Células HeLa , Células HEK293 , FemininoRESUMO
Fangchinoline (FA) is an alkaloid derived from the traditional Chinese medicine Fangji. Numerous studies have shown that FA has a toxic effect on various cancer cells, but little is known about its toxic effects on germ cells, especially oocytes. In this study, we investigated the effects of FA on mouse oocyte maturation and its potential mechanisms. Our results showed that FA did not affect meiosis resumption but inhibited the first polar body extrusion. This inhibition is not due to abnormalities at the organelle level, such as chromosomes and mitochondrial, which was proved by detection of DNA damage and reactive oxygen species. Further studies revealed that FA arrested the oocyte at the metaphase I stage, and this arrest was not caused by abnormal kinetochore-microtubule attachment or spindle assembly checkpoint activation. Instead, FA inhibits the activity of anaphase-promoting complexes (APC/C), as evidenced by the inhibition of CCNB1 degeneration. The decreased activity of APC/C may be due to a reduction in CDC25B activity as indicated by the high phosphorylation level of CDC25B (Ser323). This may further enhance Maturation-Promoting Factor (MPF) activity, which plays a critical role in meiosis. In conclusion, our study suggests that the metaphase I arrest caused by FA may be due to abnormalities in MPF and APC/C activity.
Assuntos
Benzilisoquinolinas , Fator Promotor de Maturação , Meiose , Mesotelina , Oócitos , Animais , Meiose/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Feminino , Benzilisoquinolinas/farmacologia , Fator Promotor de Maturação/metabolismo , Camundongos , Fosfatases cdc25/metabolismo , Fosfatases cdc25/genética , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Camundongos Endogâmicos ICR , Espécies Reativas de Oxigênio/metabolismo , Dano ao DNA/efeitos dos fármacos , Ciclina B1/metabolismo , Ciclina B1/genéticaRESUMO
Paclitaxel induces multipolar spindles at clinically relevant doses but does not substantially increase mitotic indices. Paclitaxel's anti-cancer effects are hypothesized to occur by promoting chromosome mis-segregation on multipolar spindles leading to apoptosis, necrosis and cyclic-GMP-AMP Synthase-Stimulator of Interferon Genes (cGAS-STING) pathway activation in daughter cells, leading to secretion of type I interferon (IFN) and immunogenic cell death. Eribulin and vinorelbine have also been reported to cause increases in multipolar spindles in cancer cells. Recently, suppression of Anaphase-Promoting Complex/Cyclosome-Cell Division Cycle 20 (APC/C-CDC20) activity using CRISPR/Cas9 mutagenesis has been reported to increase sensitivity to Kinesin Family 18a (KIF18a) inhibition, which functions to suppress multipolar mitotic spindles in cancer cells. We propose that a way to enhance the effectiveness of anti-cancer agents that increase multipolar spindles is by suppressing the APC/C-CDC20 to delay, but not block, anaphase entry. Delaying anaphase entry in genomically unstable cells may enhance multipolar spindle-induced cell death. In genomically stable healthy human cells, delayed anaphase entry may suppress the level of multipolar spindles induced by anti-cancer drugs and lower mitotic cytotoxicity. We outline specific combinations of molecules to investigate that may achieve the goal of enhancing the effectiveness of anti-cancer agents.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase , Antineoplásicos , Fuso Acromático , Humanos , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Antineoplásicos/farmacologia , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/metabolismo , Proteínas Cdc20/metabolismo , Proteínas Cdc20/genética , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Mitose/efeitos dos fármacosRESUMO
BACKGROUND: In addition to functioning as a precise monitoring mechanism in cell cycle, the anaphase-promoting complex/cyclosome (APC/C) is reported to be involved in regulating multiple metabolic processes by facilitating the ubiquitin-mediated degradation of key enzymes. Fatty acid oxidation is a metabolic pathway utilized by tumor cells that is crucial for malignant progression; however, its association with APC/C remains to be explored. METHODS: Cell cycle synchronization, immunoblotting, and propidium iodide staining were performed to investigate the carnitine palmitoyltransferase 1 C (CPT1C) expression manner. Proximity ligation assay and co-immunoprecipitation were performed to detect interactions between CPT1C and APC/C. Flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium, inner salt (MTS) assays, cell-scratch assays, and transwell assays and xenograft transplantation assays were performed to investigate the role of CPT1C in tumor progression in vitro and in vivo. Immunohistochemistry was performed on tumor tissue microarray to evaluate the expression levels of CPT1C and explore its potential clinical value. RESULTS: We identified CPT1C as a novel APC/C substrate. CPT1C protein levels exhibited cell cycle-dependent fluctuations, peaking at the G1/S boundary. Elevated CPT1C accelerated the G1/S transition, facilitating tumor cell proliferation in vitro and in vivo. Furthermore, CPT1C enhanced fatty acid utilization, upregulated ATP levels, and decreased reactive oxygen species levels, thereby favoring cell survival in a harsh metabolic environment. Clinically, high CPT1C expression correlated with poor survival in patients with esophageal squamous cell carcinoma. CONCLUSIONS: Overall, our results revealed a novel interplay between fatty acid utilization and cell cycle machinery in tumor cells. Additionally, CPT1C promoted tumor cell proliferation and survival by augmenting cellular ATP levels and preserving redox homeostasis, particularly under metabolic stress. Therefore, CPT1C could be an independent prognostic indicator in esophageal squamous cell carcinoma.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase , Carnitina O-Palmitoiltransferase , Carnitina O-Palmitoiltransferase/metabolismo , Carnitina O-Palmitoiltransferase/genética , Humanos , Animais , Linhagem Celular Tumoral , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/genética , Metabolismo Energético/genética , Regulação para Cima , Progressão da Doença , Proliferação de Células , Camundongos Nus , Camundongos , Feminino , Masculino , Fase S , Camundongos Endogâmicos BALB CRESUMO
The anaphase-promoting complex/cyclosome (APC/C) is a critical and tightly regulated E3 ligase that orchestrates the cellular life cycle by controlling the degradation of cell cycle regulators. An intriguing feature of this complex is an autoinhibition mechanism: an intrinsically disordered loop domain, Apc1-300L, blocks Cdc20 coactivator binding, yet phosphorylation of Apc1-300L counteracts this autoinhibition. Many such disordered loops within APC/C remain unexplored. Our systematic analysis of loop-deficient APC/C mutants uncovered a pivotal role for Apc8's C-terminal loop (Apc8-L) in mitotic activation. Apc8-L directly recruits the CDK adaptor protein, Xe-p9/Cks2, positioning the Xe-p9-CDK-CycB complex near Apc1-300L. This stimulates the phosphorylation and removal of Apc1-300L, prompting the formation of active APC/CCdc20. Strikingly, without both Apc8-L and Apc3-L, the APC/C is rendered inactive during mitosis, highlighting Apc8-L's synergistic role with other loops and kinases. This study broadens our understanding of the intricate dynamics in APC/C regulation and provides insights on the regulation of macromolecular complexes.
Assuntos
Mitose , Animais , Feminino , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Subunidade Apc8 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas Cdc20/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Fosforilação , Ligação Proteica , Domínios Proteicos , Xenopus laevisRESUMO
Induced oncoproteins degradation provides an attractive anti-cancer modality. Activation of anaphase-promoting complex (APC/CCDH1) prevents cell-cycle entry by targeting crucial mitotic proteins for degradation. Phosphorylation of its co-activator CDH1 modulates the E3 ligase activity, but little is known about its regulation after phosphorylation and how to effectively harness APC/CCDH1 activity to treat cancer. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1)-catalyzed phosphorylation-dependent cis-trans prolyl isomerization drives tumor malignancy. However, the mechanisms controlling its protein turnover remain elusive. Through proteomic screens and structural characterizations, we identify a reciprocal antagonism of PIN1-APC/CCDH1 mediated by domain-oriented phosphorylation-dependent dual interactions as a fundamental mechanism governing mitotic protein stability and cell-cycle entry. Remarkably, combined PIN1 and cyclin-dependent protein kinases (CDKs) inhibition creates a positive feedback loop of PIN1 inhibition and APC/CCDH1 activation to irreversibly degrade PIN1 and other crucial mitotic proteins, which force permanent cell-cycle exit and trigger anti-tumor immunity, translating into synergistic efficacy against triple-negative breast cancer.
Assuntos
Proteínas de Ciclo Celular , Proteômica , Ciclo Celular/fisiologia , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Fosforilação , Estabilidade Proteica , Peptidilprolil Isomerase de Interação com NIMA/genética , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , MitoseRESUMO
Cell division cycle 23 (CDC23) is a component of the tetratricopeptide repeat (TPR) subunit in the anaphase-promoting complex or cyclosome (APC/C) complex, which participates in the regulation of mitosis in eukaryotes. However, the regulatory model and mechanism by which the CDC23 gene regulates muscle production in pigs are largely unknown. In this study, we investigated the expression of CDC23 in pigs, and the results indicated that CDC23 is widely expressed in various tissues and organs. In vitro cell experiments have demonstrated that CDC23 promotes the proliferation of myoblasts, as well as significantly positively regulating the differentiation of skeletal muscle satellite cells. In addition, Gene Set Enrichment Analysis (GSEA) revealed a significant downregulation of the cell cycle pathway during the differentiation process of skeletal muscle satellite cells. The protein-protein interaction (PPI) network showed a high degree of interaction between genes related to the cell cycle pathway and CDC23. Subsequently, in differentiated myocytes induced after overexpression of CDC23, the level of CDC23 exhibited a significant negative correlation with the expression of key factors in the cell cycle pathway, suggesting that CDC23 may be involved in the inhibition of the cell cycle signaling pathway in order to promote the differentiation process. In summary, we preliminarily determined the function of CDC23 with the aim of providing new insights into molecular regulation during porcine skeletal muscle development.
Assuntos
Músculo Esquelético , Células Satélites de Músculo Esquelético , Animais , Ciclossomo-Complexo Promotor de Anáfase , Células Musculares , SuínosRESUMO
Cell cycle control relies on a delicate balance of phosphorylation with CDK1 and phosphatases like PP1 and PP2A-B55. Yet, identifying the primary substrate responsible for cell cycle oscillations remains a challenge. We uncover the pivotal role of phospho-regulation in the anaphase-promoting complex/cyclosome (APC/C), particularly through the Apc1-loop300 domain (Apc1-300L), orchestrated by CDK1 and PP2A-B55. Premature activation of PP2A-B55 during mitosis, induced by Greatwall kinase depletion, leads to Apc1-300L dephosphorylation, stalling APC/C activity and delaying Cyclin B degradation. This effect can be counteracted using the B55-specific inhibitor pEnsa or by removing Apc1-300L. We also show Cdc20's dynamic APC/C interaction across cell cycle stages, but dephosphorylation of Apc1-300L specifically inhibits further Cdc20 recruitment. Our study underscores APC/C's central role in cell cycle oscillation, identifying it as a primary substrate regulated by the CDK-PP2A partnership.
Assuntos
Proteína Quinase CDC2 , Ciclo Celular , Proteína Fosfatase 2 , Animais , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteína Quinase CDC2/metabolismo , Proteínas Cdc20/metabolismo , Mitose , Fosforilação , Proteína Fosfatase 2/metabolismo , Células Sf9 , XenopusRESUMO
Whole-genome duplication (WGD) is a frequent event in cancer evolution that fuels chromosomal instability. WGD can result from mitotic errors or endoreduplication, yet the molecular mechanisms that drive WGD remain unclear. Here, we use live single-cell analysis to characterize cell-cycle dynamics upon aberrant Ras-ERK signaling. We find that sustained ERK signaling in human cells leads to reactivation of the APC/C in G2, resulting in tetraploid G0-like cells that are primed for WGD. This process is independent of DNA damage or p53 but dependent on p21. Transcriptomics analysis and live-cell imaging showed that constitutive ERK activity promotes p21 expression, which is necessary and sufficient to inhibit CDK activity and which prematurely activates the anaphase-promoting complex (APC/C). Finally, either loss of p53 or reduced ERK signaling allowed for endoreduplication, completing a WGD event. Thus, sustained ERK signaling-induced G2 cell cycle exit represents an alternative path to WGD.
Assuntos
Sistema de Sinalização das MAP Quinases , Proteína Supressora de Tumor p53 , Humanos , Sistema de Sinalização das MAP Quinases/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Genoma Humano , Duplicação Gênica , Fase G2/genética , Dano ao DNA/genéticaRESUMO
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.
Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/genética , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas Cdc20/genética , Proteínas Cdc20/metabolismo , Proteínas de Ciclo Celular/metabolismo , Mitose , Fuso Acromático/metabolismoRESUMO
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.
Assuntos
Pontos de Checagem da Fase M do Ciclo Celular , Meiose , Animais , Feminino , Camundongos , Ciclossomo-Complexo Promotor de Anáfase/genética , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Oócitos/metabolismo , Ubiquitinas/metabolismoRESUMO
Meiosis is a complex variant of the mitotic cell cycle, and as such relies on many of the same proteins involved in mitosis, but utilizes these in novel ways. As in mitosis, Cdk1 and its cyclin partners, Cyclin A, B, and B3 are required at multiple steps in meiosis. Here, we study the effect of stabilized forms of the three mitotic cyclins to study the consequences of failure to degrade the cyclins in meiosis. We find that stabilized Cyclin B3 promotes ectopic microtubule polymerization throughout the egg, dependent on APC/C activity and apparently due to the consequent destruction of Cyclin A and Cyclin B. We present data that suggests CycB, and possibly CycA, can also promote APC/C activity at specific stages of meiosis. We also present evidence that in meiosis APC/CCort and APC/CFzy are able to target Cyclin B via a novel degron. Overall, our findings highlight the distinct functions of the three mitotic Cdk-cyclin complexes in meiosis.
Assuntos
Ciclina B , Ciclinas , Proteínas de Drosophila , Meiose , Mitose , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Ciclina B/metabolismo , Ciclina B/genética , Ciclinas/metabolismo , Ciclinas/genética , Ciclina A/metabolismo , Drosophila/metabolismo , Drosophila/genética , Microtúbulos/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genéticaRESUMO
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.
Assuntos
Triticum , Ubiquitina-Proteína Ligases , Ciclossomo-Complexo Promotor de Anáfase/genética , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Triticum/genética , Triticum/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/genética , Ubiquitina/genéticaRESUMO
Alterations in cell cycle regulation contribute to Zika virus (ZIKV)-associated pathogenesis and may have implications for the development of therapeutic avenues. As a matter of fact, ZIKV alters cell cycle progression at multiple stages, including G1, S, G2, and M phases. During a cell cycle, the progression of mitosis is particularly controlled to avoid any abnormalities in cell division. In this regard, the critical metaphase-anaphase transition is triggered by the activation of anaphase-promoting complex/cyclosome (APC/C) by its E3 ubiquitin ligase subunit Cdc20. Cdc20 recognizes substrates by interacting with a destruction box motif (D-box). Recently, the ZIKV nonstructural protein 5 (NS5), one of the most highly conserved flavivirus proteins, has been shown to localize to the centrosome in each pole and to spindle fibers during mitosis. Inducible expression of NS5 reveals an interaction of this viral factor with centrosomal proteins leading to an increase in the time required to complete mitosis. By analyzing the NS5 sequence, we discovered the presence of a D-box. Taken together, these data support the idea that, in addition to its role in viral replication, NS5 plays a critical role in the control of the cell cycle of infected cells and, more specifically, in the regulation of the mitotic spindle. Here we propose that the NS5 protein may interfere with the metaphase-anaphase progression, and thus cause the observed delay in mitosis via the regulation of APC/C.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase , Mitose , Proteínas não Estruturais Virais , Infecção por Zika virus , Zika virus , Humanos , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas Cdc20/metabolismo , Ciclo Celular , Centrossomo/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Zika virus/fisiologia , Zika virus/metabolismo , Infecção por Zika virus/metabolismo , Infecção por Zika virus/virologia , Infecção por Zika virus/patologiaRESUMO
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.
Assuntos
Proteínas Cdc20 , Pontos de Checagem da Fase M do Ciclo Celular , Fuso Acromático , Ciclossomo-Complexo Promotor de Anáfase/genética , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas Cdc20/química , Proteínas Cdc20/genética , Proteínas Cdc20/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/genética , Fuso Acromático/genética , Fuso Acromático/metabolismo , Transdução de Sinais , HumanosRESUMO
Sine oculis homeobox homolog 1 (Six1) is a developmentally important transcription factor that regulates cellular proliferation, apoptosis, and dissemination during embryogenesis. Six1 overexpression as reported in multiple cancers modulates expression of a repertoire of its target genes causing an increase in proliferation, metastasis and survival of cancer cells. Six1 exists as a cell cycle regulated nuclear phosphoprotein and its cellular turnover is regulated by APC/C (Anaphase promoting complex / Cyclosome) complex mediated proteolysis. However, the kinases that regulate Six1 proteolysis have not been identified and the mechanistic details that cause its overproduction in various cancers are lacking. Here, we report that Six1 is a physiological GSK3ß substrate. GSK3ß interacts with Six1 and phosphorylates it at Ser221 within the conserved consensus sequence in its carboxy terminus. Using pharmacological inhibition, siRNA mediated knockdown and protein overexpression of GSK3ß; we show that GSK3ß regulates Six1 protein stability. Pulse chase analysis of Six1 revealed that GSK3ß regulates its ubiquitin proteolysis such that Six1 phosphomimicking mutant (Six1S221E) for Ser221 site had dramatically increased half-life than its phosphodeficient (Six1S221A) and wild type variants. Furthermore, we demonstrate that GSK3ß rescues Six1 from APC dependent proteolysis by regulating its binding with APC/C co-activator protein Cdh1. Importantly, strong positive correlation exists between GSK3ß and Six1 protein levels throughout the cell cycle and in multiple cancers indicating that GSK3ß activation may in part contribute to Six1 overproduction in a subset of human cancers.
Assuntos
Proteínas de Ciclo Celular , Fatores de Transcrição , Humanos , Glicogênio Sintase Quinase 3 beta , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Proteínas Cdh1/metabolismoRESUMO
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.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase , Neoplasias , Humanos , Ciclossomo-Complexo Promotor de Anáfase/genética , Dineínas , Cinesinas/genética , Cinetocoros , Mitose , Neoplasias/genéticaRESUMO
The mammalian cell cycle alternates between two phases - S-G2-M with high levels of A- and B-type cyclins (CycA and CycB, respectively) bound to cyclin-dependent kinases (CDKs), and G1 with persistent degradation of CycA and CycB by an activated anaphase promoting complex/cyclosome (APC/C) bound to Cdh1 (also known as FZR1 in mammals; denoted APC/C:Cdh1). Because CDKs phosphorylate and inactivate Cdh1, these two phases are mutually exclusive. This 'toggle switch' is flipped from G1 to S by cyclin-E bound to a CDK (CycE:CDK), which is not degraded by APC/C:Cdh1, and from M to G1 by Cdc20-bound APC/C (APC/C:Cdc20), which is not inactivated by CycA:CDK or CycB:CDK. After flipping the switch, cyclin E is degraded and APC/C:Cdc20 is inactivated. Combining mathematical modelling with single-cell timelapse imaging, we show that dysregulation of CycB:CDK disrupts strict alternation of the G1-S and M-G1 switches. Inhibition of CycB:CDK results in Cdc20-independent Cdh1 'endocycles', and sustained activity of CycB:CDK drives Cdh1-independent Cdc20 endocycles. Our model provides a mechanistic explanation for how whole-genome doubling can arise, a common event in tumorigenesis that can drive tumour evolution.
Assuntos
Proteínas de Ciclo Celular , Ciclinas , Animais , Ciclo Celular , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Mitose , Proteínas Cdc20/metabolismo , Mamíferos/metabolismoRESUMO
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.