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Molecular Biology of the Cell Apr 2020E2F8 is a transcriptional repressor that antagonizes E2F1 at the crossroads of the cell cycle, apoptosis, and cancer. Previously, we discovered that E2F8 is a direct...
E2F8 is a transcriptional repressor that antagonizes E2F1 at the crossroads of the cell cycle, apoptosis, and cancer. Previously, we discovered that E2F8 is a direct target of the APC/C ubiquitin ligase. Nevertheless, it remains unknown how E2F8 is dynamically controlled throughout the entirety of the cell cycle. Here, using newly developed human cell-free systems that recapitulate distinct inter-mitotic and G1 phases and a continuous transition from prometaphase to G1, we reveal an interlocking dephosphorylation switch coordinating E2F8 degradation with mitotic exit and the activation of APC/C. Further, we uncover differential proteolysis rates for E2F8 at different points within G1 phase, accounting for its accumulation in late G1 while APC/C is still active. Finally, we demonstrate that the F-box protein Cyclin F regulates E2F8 in G2-phase. Altogether, our data define E2F8 regulation throughout the cell cycle, illuminating an extensive coordination between phosphorylation, ubiquitination and transcription in mammalian cell cycle.
Topics: Amino Acid Motifs; Apc1 Subunit, Anaphase-Promoting Complex-Cyclosome; Cell Cycle; Cell-Free System; Cyclins; E2F1 Transcription Factor; G1 Phase; G2 Phase; HeLa Cells; Humans; Mitosis; Phosphorylation; Protein Processing, Post-Translational; Proteolysis; Recombinant Proteins; Repressor Proteins; Ubiquitination
PubMed: 31995441
DOI: 10.1091/mbc.E19-12-0725 -
European Review For Medical and... May 2022We have previously reported the novel off-target microtubules destabilizing activity of SB225002, a compound that was originally designed as a selective and potent IL-8...
OBJECTIVE
We have previously reported the novel off-target microtubules destabilizing activity of SB225002, a compound that was originally designed as a selective and potent IL-8 receptor B antagonist. In the present study we investigated the reversibility of SB225002 antimitotic effect and provided additional mechanistic insights underlying cell death induction in SW480 human colorectal adenocarcinoma cells.
MATERIALS AND METHODS
Mitotically arrested cells by SB225002 treatment were isolated by shake-off, and their identity was verified by both flow cytometry and immunoblotting. The reversibility of SB225002 antimitotic effects was investigated by flow cytometry and immunoblotting. Prometaphase arrested cells were imaged via indirect immunofluorescence and confocal microscopy. Activation of CHK1 in mitotically arrested cells was assessed by immunoblotting, and the relationship between CHK1 and mitotic arrest was examined via siRNA-mediated knockdown of CHK1. JNK signaling was evaluated via immunoblotting as well as pharmacological inhibition, followed by flow cytometry. The role of reactive oxygen species (ROS) in cytotoxicity was evaluated by ROS scavenging and flow cytometry.
RESULTS
Following SB225002 washout, the mitotic checkpoint was abrogated, and cell cycle perturbations were gradually restored with induction of cell death. Mechanistically, CHK1 checkpoint was activated by SB225002 and occurred downstream of the mitotic checkpoint. In addition, SB225002 activated JNK signaling which contributed to cell death and restrained polyploidy. Furthermore, SB225002 increased intracellular ROS which played a role in mediating SB225002 cytotoxicity.
CONCLUSIONS
Findings of the present study warrants further development of SB225002 as a lead compound that uniquely targets microtubules dynamics and IL-8 signaling.
Topics: Antimitotic Agents; Humans; Microtubules; Phenylurea Compounds; Reactive Oxygen Species; Receptors, Interleukin-8
PubMed: 35647855
DOI: 10.26355/eurrev_202205_28869 -
BioRxiv : the Preprint Server For... Dec 2023To ensure genomic fidelity a series of spatially and temporally coordinated events are executed during prometaphase of mitosis, including bipolar spindle formation,...
To ensure genomic fidelity a series of spatially and temporally coordinated events are executed during prometaphase of mitosis, including bipolar spindle formation, chromosome attachment to spindle microtubules at kinetochores, the correction of erroneous kinetochore-microtubule (k-MT) attachments, and chromosome congression to the spindle equator. Cyclin A/Cdk1 kinase plays a key role in destabilizing k-MT attachments during prometaphase to promote correction of erroneous k-MT attachments. However, it is unknown if Cyclin A/Cdk1 kinase regulates other events during prometaphase. Here, we investigate additional roles of Cyclin A/Cdk1 in prometaphase by using an siRNA knockdown strategy to deplete endogenous Cyclin A from human cells. We find that depleting Cyclin A significantly extends mitotic duration, specifically prometaphase, because chromosome alignment is delayed. Unaligned chromosomes display erroneous monotelic, syntelic, or lateral k-MT attachments suggesting that bioriented k-MT attachment formation is delayed in the absence of Cyclin A. Mechanistically, chromosome alignment is likely impaired because the localization of the kinetochore proteins BUB1 kinase, KNL1, and MPS1 kinase are reduced in Cyclin A-depleted cells. Moreover, we find that Cyclin A promotes BUB1 kinetochore localization independently of its role in destabilizing k-MT attachments. Thus, Cyclin A/Cdk1 facilitates chromosome alignment during prometaphase to support timely mitotic progression.
PubMed: 38187612
DOI: 10.1101/2023.12.21.572788 -
Cell Chemical Biology Mar 2020Polo-like kinase 1 has hundreds of substrates and multiple functions that operate within the ∼60 min of mitosis. Herein, we describe a chemical-genetic system that...
Polo-like kinase 1 has hundreds of substrates and multiple functions that operate within the ∼60 min of mitosis. Herein, we describe a chemical-genetic system that allows particular substrates to be "toggled" into or out of chemical control using engineered phosphoacceptor selectivity. Biochemical assays and phosphoproteomic analysis of mitotic cell extracts showed that Plk1 (L197F) and Plk1 (L197S/L211A) selectively phosphorylate Ser and Thr, respectively. Plk1 but not Plk1 sustains mitotic progression to anaphase, affording the opportunity to toggle substrate residues between Ser and Thr to place them under chemical control. Using this system, we evaluated Kif2b, a known substrate of Plk1 that regulates chromosome alignment. Toggling Ser to Thr on Kif2b places these phosphorylation sites under reversible chemical control, as indicated by a sharp increase in the frequency of misaligned chromosomes and prometaphase arrest. Thus, we demonstrate the ability to chemically control a single substrate by a genetic Ser/Thr toggle.
Topics: Cell Cycle Proteins; Humans; Mitosis; Phosphorylation; Protein Engineering; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Serine; Substrate Specificity; Threonine; Polo-Like Kinase 1
PubMed: 32017920
DOI: 10.1016/j.chembiol.2020.01.007 -
Cell Reports Jul 2021Stable transmission of genetic material during cell division requires accurate chromosome segregation. PLK1 dynamics at kinetochores control establishment of correct...
Stable transmission of genetic material during cell division requires accurate chromosome segregation. PLK1 dynamics at kinetochores control establishment of correct kinetochore-microtubule attachments and subsequent silencing of the spindle checkpoint. However, the regulatory mechanism responsible for PLK1 activity in prometaphase has not yet been affirmatively identified. Here we identify Apolo1, which tunes PLK1 activity for accurate kinetochore-microtubule attachments. Apolo1 localizes to kinetochores during early mitosis, and suppression of Apolo1 results in misaligned chromosomes. Using the fluorescence resonance energy transfer (FRET)-based PLK1 activity reporter, we found that Apolo1 sustains PLK1 kinase activity at kinetochores for accurate attachment during prometaphase. Apolo1 is a cognate substrate of PLK1, and the phosphorylation enables PP1γ to inactivate PLK1 by dephosphorylation. Mechanistically, Apolo1 constitutes a bridge between kinase and phosphatase, which governs PLK1 activity in prometaphase. These findings define a previously uncharacterized feedback loop by which Apolo1 provides fine-tuning for PLK1 to guide chromosome segregation in mitosis.
Topics: Amino Acid Motifs; Amino Acid Sequence; Cell Cycle Proteins; Chromosome Segregation; Feedback, Physiological; HEK293 Cells; HeLa Cells; Humans; Kinetochores; Mitosis; Phosphoprotein Phosphatases; Phosphorylation; Phosphoserine; Protein Binding; Protein Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins; Polo-Like Kinase 1
PubMed: 34260926
DOI: 10.1016/j.celrep.2021.109343 -
ELife Aug 2022Btg3-associated nuclear protein (Banp) was originally identified as a nuclear matrix-associated region (MAR)-binding protein and it functions as a tumor suppressor. At...
Btg3-associated nuclear protein (Banp) was originally identified as a nuclear matrix-associated region (MAR)-binding protein and it functions as a tumor suppressor. At the molecular level, Banp regulates transcription of metabolic genes via a CGCG-containing motif called the Banp motif. However, its physiological roles in embryonic development are unknown. Here, we report that Banp is indispensable for the DNA damage response and chromosome segregation during mitosis. Zebrafish mutants show mitotic cell accumulation and apoptosis in developing retina. We found that DNA replication stress and tp53-dependent DNA damage responses were activated to induce apoptosis in mutants, suggesting that Banp is required for regulation of DNA replication and DNA damage repair. Furthermore, consistent with mitotic cell accumulation, chromosome segregation was not smoothly processed from prometaphase to anaphase in morphants, leading to a prolonged M-phase. Our RNA- and ATAC-sequencing identified 31 candidates for direct Banp target genes that carry the Banp motif. Interestingly, a DNA replication fork regulator, and two chromosome segregation regulators, and , are included in this list. Thus, Banp directly regulates transcription of for recovery from DNA replication stress, and and for chromosome segregation during mitosis. Our findings provide the first in vivo evidence that Banp is required for cell-cycle progression and cell survival by regulating DNA damage responses and chromosome segregation during mitosis.
Topics: Animals; Cell Cycle; Chromosome Segregation; Chromosomes; DNA Damage; Mitosis; Nuclear Proteins; Retina; Zebrafish
PubMed: 35942692
DOI: 10.7554/eLife.74611 -
Journal of Proteome Research Jul 2021The spindle assembly checkpoint (SAC) is critical for sensing defective microtubule-kinetochore attachments and tension across the kinetochore and functions to arrest...
The spindle assembly checkpoint (SAC) is critical for sensing defective microtubule-kinetochore attachments and tension across the kinetochore and functions to arrest cells in prometaphase to allow time to repair any errors before proceeding into anaphase. Dysregulation of the SAC leads to chromosome segregation errors that have been linked to human diseases like cancer. Although much has been learned about the composition of the SAC and the factors that regulate its activity, the proximity associations of core SAC components have not been explored in a systematic manner. Here, we have taken a BioID2-proximity-labeling proteomic approach to define the proximity protein environment for each of the five core SAC proteins BUB1, BUB3, BUBR1, MAD1L1, and MAD2L1 in mitotic-enriched populations of cells where the SAC is active. These five protein association maps were integrated to generate a SAC proximity protein network that contains multiple layers of information related to core SAC protein complexes, protein-protein interactions, and proximity associations. Our analysis validated many known SAC complexes and protein-protein interactions. Additionally, it uncovered new protein associations, including the ELYS-MAD1L1 interaction that we have validated, which lend insight into the functioning of core SAC proteins and highlight future areas of investigation to better understand the SAC.
Topics: Cell Cycle Proteins; Humans; Kinetochores; M Phase Cell Cycle Checkpoints; Protein Serine-Threonine Kinases; Proteomics; Spindle Apparatus
PubMed: 34087075
DOI: 10.1021/acs.jproteome.0c00941 -
Nature Communications Jun 2023The centromere is the chromosome region where microtubules attach during cell division. In contrast to monocentric chromosomes with one centromere, holocentric species...
The centromere is the chromosome region where microtubules attach during cell division. In contrast to monocentric chromosomes with one centromere, holocentric species usually distribute hundreds of centromere units along the entire chromatid. We assembled the chromosome-scale reference genome and analyzed the holocentromere and (epi)genome organization of the lilioid Chionographis japonica. Remarkably, each of its holocentric chromatids consists of only 7 to 11 evenly spaced megabase-sized centromere-specific histone H3-positive units. These units contain satellite arrays of 23 and 28 bp-long monomers capable of forming palindromic structures. Like monocentric species, C. japonica forms clustered centromeres in chromocenters at interphase. In addition, the large-scale eu- and heterochromatin arrangement differs between C. japonica and other known holocentric species. Finally, using polymer simulations, we model the formation of prometaphase line-like holocentromeres from interphase centromere clusters. Our findings broaden the knowledge about centromere diversity, showing that holocentricity is not restricted to species with numerous and small centromere units.
Topics: Centromere; Cell Cycle Proteins; Cell Division; Chromatids; Heterochromatin
PubMed: 37311740
DOI: 10.1038/s41467-023-38922-7 -
EMBO Reports Jun 2020The anaphase-promoting complex (APC/C) is the key E3 ubiquitin ligase which directs mitotic progression and exit by catalysing the sequential ubiquitination of specific...
The anaphase-promoting complex (APC/C) is the key E3 ubiquitin ligase which directs mitotic progression and exit by catalysing the sequential ubiquitination of specific substrates. The activity of the APC/C in mitosis is restrained by the spindle assembly checkpoint (SAC), which coordinates chromosome segregation with the assembly of the mitotic spindle. The SAC effector is the mitotic checkpoint complex (MCC), which binds and inhibits the APC/C. It is incompletely understood how the APC/C switches substrate specificity in a cell cycle-specific manner. For instance, it is unclear how in prometaphase, when APC/C activity towards cyclin B and securin is repressed by the MCC, the kinase Nek2A is ubiquitinated. Here, we combine biochemical and structural analysis with functional studies in cells to show that Nek2A is a conformational-specific binder of the APC/C-MCC complex (APC/C ) and that, in contrast to cyclin A, Nek2A can be ubiquitinated efficiently by the APC/C in conjunction with both the E2 enzymes UbcH10 and UbcH5. We propose that these special features of Nek2A allow its prometaphase-specific ubiquitination.
Topics: Anaphase-Promoting Complex-Cyclosome; Cdc20 Proteins; Cell Cycle Proteins; HeLa Cells; Humans; M Phase Cell Cycle Checkpoints; Mitosis; Prometaphase; Spindle Apparatus; Ubiquitination
PubMed: 32307883
DOI: 10.15252/embr.201949831 -
Journal of Cell Science Jun 2022Mitotic kinesin-like protein 2 (MKLP2; also known as KIF20A) is a motor protein with a well-established function in promoting cytokinesis. However, our results with...
Mitotic kinesin-like protein 2 (MKLP2; also known as KIF20A) is a motor protein with a well-established function in promoting cytokinesis. However, our results with siRNAs targeting MKLP2 and small-molecule inhibitors of MKLP2 (MKLP2i) suggest that it also has a function earlier in mitosis, prior to anaphase. In this study, we provide direct evidence that MKLP2 facilitates chromosome congression in prometaphase. We employed live imaging to observe HeLa cells with fluorescently tagged histones treated with MKLP2i and discovered a pronounced chromosome congression defect. We show that MKLP2 facilitates error correction, as inhibited cells have a significant increase in unstable, syntelic kinetochore-microtubule attachments. We find that the aberrant attachments are accompanied by elevated Aurora kinase (A and B) activity and phosphorylation of the downstream target HEC1 (also known as NDC80) at Ser55. Finally, we show that MKLP2 inhibition results in aneuploidy, confirming that MKLP2 safeguards cells against chromosomal instability. This article has an associated First Person interview with the first author of the paper.
Topics: Aurora Kinase B; Chromosome Segregation; Chromosomes; HeLa Cells; Humans; Kinesins; Kinetochores; Microtubules; Mitosis; Spindle Apparatus
PubMed: 35638575
DOI: 10.1242/jcs.259560