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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 -
Oncotarget Jan 2018Cis-trimethoxy resveratrol (cis-3M-RES) induced dose-dependent cytotoxicity and apoptotic DNA fragmentation in Jurkat T cell clones (JT/Neo); however, it induced only...
Cis-trimethoxy resveratrol (cis-3M-RES) induced dose-dependent cytotoxicity and apoptotic DNA fragmentation in Jurkat T cell clones (JT/Neo); however, it induced only cytostasis in BCL-2-overexpressing cells (JT/BCL-2). Treatment with 0.25 μM cis-3M-RES induced G/M arrest, BAK activation, Δψm loss, caspase-9 and caspase-3 activation, and poly (ADP-ribose) polymerase (PARP) cleavage in JT/Neo cells time-dependently but did not induce these events, except G/M arrest, in JT/BCL-2 cells. Moreover, cis-3M-RES induced CDK1 activation, BCL-2 phosphorylation at Ser-70, MCL-1 phosphorylation at Ser-159/Thr-163, and BIM (BIM and BIM) phosphorylation irrespective of BCL-2 overexpression. Enforced G/S arrest by using a G/S blocker aphidicolin completely inhibited cis-3M-RES-induced apoptotic events. Cis-3M-RES-induced phosphorylation of BCL-2 family proteins and mitochondrial apoptotic events were suppressed by a validated CDK1 inhibitor RO3306. Immunofluorescence microscopy showed that cis-3M-RES induced mitotic spindle defects and prometaphase arrest. The rate of intracellular polymeric tubulin to monomeric tubulin decreased markedly by cis-3M-RES (0.1-1.0 μM). Wild-type Jurkat clone A3, FADD-deficient Jurkat clone I2.1, and caspase-8-deficient Jurkat clone I9.2 exhibited similar susceptibilities to the cytotoxicity of cis-3M-RES, excluding contribution of the extrinsic death receptor-dependent pathway to the apoptosis. IC values of cis-3M-RES against Jurkat E6.1, U937, HL-60, and HeLa cells were 0.07-0.17 μM, whereas those against unstimulated human peripheral T cells and phytohaemagglutinin A-stimulated peripheral T cells were >10.0 and 0.23 μM, respectively. These results indicate that the antitumor activity of cis-3M-RES is mediated by microtubule damage, and subsequent prometaphase arrest and prolonged CDK1 activation that cause BAK-mediated mitochondrial apoptosis, and suggest that cis-3M-RES is a promising agent to treat leukemia.
PubMed: 29435156
DOI: 10.18632/oncotarget.23576 -
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 -
The Journal of Cell Biology Mar 2018The mitotic spindle checkpoint delays anaphase onset in the presence of unattached kinetochores, and efficient checkpoint signaling requires kinetochore localization of...
The mitotic spindle checkpoint delays anaphase onset in the presence of unattached kinetochores, and efficient checkpoint signaling requires kinetochore localization of the Rod-ZW10-Zwilch (RZZ) complex. In the present study, we show that human Chmp4c, a protein involved in membrane remodeling, localizes to kinetochores in prometaphase but is reduced in chromosomes aligned at the metaphase plate. Chmp4c promotes stable kinetochore-microtubule attachments and is required for proper mitotic progression, faithful chromosome alignment, and segregation. Depletion of Chmp4c diminishes localization of RZZ and Mad1-Mad2 checkpoint proteins to prometaphase kinetochores and impairs mitotic arrest when microtubules are depolymerized by nocodazole. Furthermore, Chmp4c binds to ZW10 through a small C-terminal region, and constitutive Chmp4c kinetochore targeting causes a ZW10-dependent checkpoint metaphase arrest. In addition, Chmp4c spindle functions do not require endosomal sorting complex required for transport-dependent membrane remodeling. These results show that Chmp4c regulates the mitotic spindle checkpoint by promoting localization of the RZZ complex to unattached kinetochores.
Topics: Cell Cycle Checkpoints; Cell Cycle Proteins; Endosomal Sorting Complexes Required for Transport; HeLa Cells; Humans; Kinetochores; Mad2 Proteins; Nuclear Proteins; Signal Transduction; Spindle Apparatus
PubMed: 29362225
DOI: 10.1083/jcb.201709005 -
Genome Research Feb 2019CCCTC-binding factor (CTCF) plays a key role in the formation of topologically associating domains (TADs) and loops in interphase. During mitosis TADs are absent, but...
CCCTC-binding factor (CTCF) plays a key role in the formation of topologically associating domains (TADs) and loops in interphase. During mitosis TADs are absent, but how TAD formation is dynamically controlled during the cell cycle is not known. Several contradicting observations have been made regarding CTCF binding to mitotic chromatin using both genomics- and microscopy-based techniques. Here, we have used four different assays to address this debate. First, using 5C, we confirmed that TADs and CTCF loops are readily detected in interphase, but absent during prometaphase. Second, ATAC-seq analysis showed that CTCF sites display greatly reduced accessibility and lose the CTCF footprint in prometaphase, suggesting loss of CTCF binding and rearrangement of the nucleosomal array around the binding motif. In contrast, transcription start sites remain accessible in prometaphase, although adjacent nucleosomes can also become repositioned and occupy at least a subset of start sites during mitosis. Third, loss of site-specific CTCF binding was directly demonstrated using CUT&RUN. Histone modifications and histone variants are maintained in mitosis, suggesting a role in bookmarking of active CTCF sites. Finally, live-cell imaging, fluorescence recovery after photobleaching, and single molecule tracking showed that almost all CTCF chromatin binding is lost in prometaphase. Combined, our results demonstrate loss of CTCF binding to CTCF sites during prometaphase and rearrangement of the chromatin landscape around CTCF motifs. This, combined with loss of cohesin, would contribute to the observed loss of TADs and CTCF loops during mitosis and reveals that CTCF sites, key architectural -elements, display cell cycle stage-dependent dynamics in factor binding and nucleosome positioning.
Topics: Binding Sites; CCCTC-Binding Factor; Cell Cycle; Cells, Cultured; Chromatin; HeLa Cells; Histone Code; Humans; Interphase; Mitosis; Nucleosomes; Nucleotide Motifs; Prometaphase; Transcription Initiation Site
PubMed: 30655336
DOI: 10.1101/gr.241547.118 -
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 -
The Journal of Cell Biology Oct 2017Cyclin A2 is a crucial mitotic Cdk regulatory partner that coordinates entry into mitosis and is then destroyed in prometaphase within minutes of nuclear envelope...
Cyclin A2 is a crucial mitotic Cdk regulatory partner that coordinates entry into mitosis and is then destroyed in prometaphase within minutes of nuclear envelope breakdown. The role of cyclin A2 in female meiosis and its dynamics during the transition from meiosis I (MI) to meiosis II (MII) remain unclear. We found that cyclin A2 decreases in prometaphase I but recovers after the first meiotic division and persists, uniquely for metaphase, in MII-arrested oocytes. Conditional deletion of cyclin A2 from mouse oocytes has no discernible effect on MI but leads to disrupted MII spindles and increased merotelic attachments. On stimulation of exit from MII, there is a dramatic increase in lagging chromosomes and an inhibition of cytokinesis. These defects are associated with an increase in microtubule stability in MII spindles, suggesting that cyclin A2 mediates the fidelity of MII by maintaining microtubule dynamics during the rapid formation of the MII spindle.
Topics: Animals; Chromosomes, Mammalian; Cyclin A2; Kinetochores; Meiosis; Mice; Mice, Knockout; Microtubules
PubMed: 28819014
DOI: 10.1083/jcb.201607111 -
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