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Cell Apr 2020Protein phosphatase 2A (PP2A) enzymes can suppress tumors, but they are often inactivated in human cancers overexpressing inhibitory proteins. Here, we identify a class...
Protein phosphatase 2A (PP2A) enzymes can suppress tumors, but they are often inactivated in human cancers overexpressing inhibitory proteins. Here, we identify a class of small-molecule iHAPs (improved heterocyclic activators of PP2A) that kill leukemia cells by allosterically assembling a specific heterotrimeric PP2A holoenzyme consisting of PPP2R1A (scaffold), PPP2R5E (B56ε, regulatory), and PPP2CA (catalytic) subunits. One compound, iHAP1, activates this complex but does not inhibit dopamine receptor D2, a mediator of neurologic toxicity induced by perphenazine and related neuroleptics. The PP2A complex activated by iHAP1 dephosphorylates the MYBL2 transcription factor on Ser241, causing irreversible arrest of leukemia and other cancer cells in prometaphase. In contrast, SMAPs, a separate class of compounds, activate PP2A holoenzymes containing a different regulatory subunit, do not dephosphorylate MYBL2, and arrest tumor cells in G1 phase. Our findings demonstrate that small molecules can serve as allosteric switches to activate distinct PP2A complexes with unique substrates.
Topics: Apoptosis; Cell Cycle Proteins; Cell Line, Tumor; Enzyme Activators; G1 Phase; Humans; Multiprotein Complexes; Phenothiazines; Phosphorylation; Protein Phosphatase 2; Protein Subunits; Trans-Activators; Transcription Factors
PubMed: 32315619
DOI: 10.1016/j.cell.2020.03.051 -
Journal of Cell Science Apr 2015Nek2 isoform A (Nek2A) is a presumed substrate of the anaphase-promoting complex/cyclosome containing Cdc20 (APC/C(Cdc20)). Nek2A, like cyclin A, is degraded in mitosis...
Nek2 isoform A (Nek2A) is a presumed substrate of the anaphase-promoting complex/cyclosome containing Cdc20 (APC/C(Cdc20)). Nek2A, like cyclin A, is degraded in mitosis while the spindle checkpoint is active. Cyclin A prevents spindle checkpoint proteins from binding to Cdc20 and is recruited to the APC/C in prometaphase. We found that Nek2A and cyclin A avoid being stabilized by the spindle checkpoint in different ways. First, enhancing mitotic checkpoint complex (MCC) formation by nocodazole treatment inhibited the degradation of geminin and cyclin A, whereas Nek2A disappeared at a normal rate. Second, depleting Cdc20 effectively stabilized cyclin A but not Nek2A. Nevertheless, Nek2A destruction crucially depended on Cdc20 binding to the APC/C. Third, in contrast to cyclin A, Nek2A was recruited to the APC/C before the start of mitosis. Interestingly, the spindle checkpoint very effectively stabilized an APC/C-binding mutant of Nek2A, which required the Nek2A KEN box. Apparently, in cells, the spindle checkpoint primarily prevents Cdc20 from binding destruction motifs. Nek2A disappearance marks the prophase-to-prometaphase transition, when Cdc20, regardless of the spindle checkpoint, activates the APC/C. However, Mad2 depletion accelerated Nek2A destruction, showing that spindle checkpoint release further increases APC/C(Cdc20) catalytic activity.
Topics: Anaphase-Promoting Complex-Cyclosome; Cdc20 Proteins; Cell Line, Tumor; Cyclin A; Geminin; Humans; M Phase Cell Cycle Checkpoints; NIMA-Related Kinases; Nocodazole; Prometaphase; Prophase; Protein Serine-Threonine Kinases
PubMed: 25673878
DOI: 10.1242/jcs.163279 -
Current Biology : CB Feb 2021The precise regulation of microtubule dynamics over time and space in dividing cells is critical for several mitotic mechanisms that ultimately enable cell...
The precise regulation of microtubule dynamics over time and space in dividing cells is critical for several mitotic mechanisms that ultimately enable cell proliferation, tissue organization, and development. Astral microtubules, which extend from the centrosome toward the cell cortex, must be present for the mitotic spindle to properly orient, as well as for the faithful execution of anaphase and cytokinesis. However, little is understood about how the dynamic properties of astral microtubules are regulated spatiotemporally, or the contribution of astral microtubule dynamics to spindle positioning. The mitotic regulator Cdk1-CyclinB promotes destabilization of centrosomal microtubules and increased microtubule dynamics as cells enter mitosis, but how Cdk1 activity modulates astral microtubule stability, and whether it impacts spindle positioning, is unknown. Here, we uncover a mechanism revealing that Cdk1 destabilizes astral microtubules in prometaphase and thereby influences spindle reorientation. Phosphorylation of the EB1-dependent microtubule plus-end tracking protein GTSE1 by Cdk1 in early mitosis abolishes its interaction with EB1 and recruitment to microtubule plus ends. Loss of Cdk1 activity, or mutation of phosphorylation sites in GTSE1, induces recruitment of GTSE1 to growing microtubule plus ends in mitosis. This decreases the catastrophe frequency of astral microtubules and causes an increase in the number of long astral microtubules reaching the cell cortex, which restrains the ability of cells to reorient spindles along the long cellular axis in early mitosis. Astral microtubules thus must not only be present but also dynamic to allow the spindle to reorient, a state assisted by selective destabilization of long astral microtubules via Cdk1.
Topics: Anaphase; Animals; CDC2 Protein Kinase; Humans; Mice; Microtubule-Associated Proteins; Microtubules; Prometaphase; Protein Stability; Spindle Apparatus
PubMed: 33333009
DOI: 10.1016/j.cub.2020.11.040 -
Scientific Reports Mar 2018Faithful chromosome segregation is ensured by the establishment of bi-orientation; the attachment of sister kinetochores to the end of microtubules extending from...
Faithful chromosome segregation is ensured by the establishment of bi-orientation; the attachment of sister kinetochores to the end of microtubules extending from opposite spindle poles. In addition, kinetochores can also attach to lateral surfaces of microtubules; called lateral attachment, which plays a role in chromosome capture and transport. However, molecular basis and biological significance of lateral attachment are not fully understood. We have addressed these questions by focusing on the prometaphase rosette, a typical chromosome configuration in early prometaphase. We found that kinetochores form uniform lateral attachments in the prometaphase rosette. Many transient kinetochore components are maximally enriched, in an Aurora B activity-dependent manner, when the prometaphase rosette is formed. We revealed that rosette formation is driven by rapid poleward motion of dynein, but can occur even in its absence, through slow kinetochore movements caused by microtubule depolymerization that is supposedly dependent on kinetochore tethering at microtubule ends by CENP-E. We also found that chromosome connection to microtubules is extensively lost when lateral attachment is perturbed in cells defective in end-on attachment. Our findings demonstrate that lateral attachment is an important intermediate in bi-orientation establishment and chromosome alignment, playing a crucial role in incorporating chromosomes into the nascent spindle.
Topics: Chromosome Segregation; Dyneins; HeLa Cells; Humans; Kinetochores; Microtubules; Prometaphase; Rosette Formation; Spindle Apparatus
PubMed: 29497093
DOI: 10.1038/s41598-018-22164-5 -
Molecular Biology of the Cell Apr 2010To maintain genomic stability, chromosome architecture needs to be tightly regulated as chromosomes undergo condensation during prophase and separation during anaphase,...
To maintain genomic stability, chromosome architecture needs to be tightly regulated as chromosomes undergo condensation during prophase and separation during anaphase, but the mechanisms remain poorly understood. Here, we show that the Plk1-binding protein PICH and Plk1 kinase coordinately maintain chromosome architecture during prometaphase. PICH knockdown results in a loss of Plk1 from the chromosome arm and an increase in highly disorganized "wavy" chromosomes that exhibit an "open" or "X-shaped" configuration, consistent with a loss of chromosome arm cohesion. Such chromosome disorganization occurs with essentially no change in the localization of condensin or cohesin on chromosomes. Interestingly, the chromosome disorganization could be prevented by treatment with a topoisomerase II inhibitor ICRF-193, suggesting that the PICH-Plk1 complex normally maintains chromosome architecture in a manner that involves topoisomerase II activity. PICH knockdown does not affect initial chromosome compaction at prophase but causes anaphase DNA bridge formation and failed abscission. Our studies suggest that the PICH-Plk1 complex plays a critical role in maintaining prometaphase chromosome architecture.
Topics: Adenosine Triphosphatases; Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; Chromosomes; DNA; DNA Helicases; DNA-Binding Proteins; Diketopiperazines; HeLa Cells; Humans; Immunoprecipitation; Microscopy, Confocal; Mitosis; Models, Biological; Multiprotein Complexes; Piperazines; Prometaphase; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Trypsin; Cohesins; Polo-Like Kinase 1
PubMed: 20130082
DOI: 10.1091/mbc.e09-11-0950 -
The Journal of Biological Chemistry Jul 2012Src family tyrosine kinases (SFKs) participate in mitotic signal transduction events, including mitotic entry, cleavage furrow ingression, and cytokinesis abscission....
Src family tyrosine kinases (SFKs) participate in mitotic signal transduction events, including mitotic entry, cleavage furrow ingression, and cytokinesis abscission. Although SFKs have been shown to associate with the mitotic spindle, the role of SFKs in mitotic spindle formation remains unclear. Here, we show that c-Src promotes proper spindle orientation in early prometaphase. Src localizes close to spindle poles in a manner independent of Src kinase activity. Three-dimensional analyses showed that Src inhibition induced spindle misorientation, exhibiting a tilting spindle in early prometaphase. Spindle misorientation is frequently seen in SYF cells, which harbor triple knock-out mutations of c-Src, c-Yes, and Fyn, and reintroduction of c-Src but not Fyn into SYF cells rescued spindle misorientation. Spindle misorientation was also observed upon Src inhibition under conditions in which Aurora B was inhibited. Inducible expression of c-Src promoted a properly oriented bipolar spindle, which was suppressed by Src inhibition. Aster formation was severely inhibited in SYF cells upon Aurora B inhibition, which was rescued by reintroduction of c-Src into SYF cells. Furthermore, reintroduction of c-Src facilitated microtubule regrowth from cold-induced depolymerization and accelerated M phase progression. These results suggest that c-Src is involved in spindle orientation through centrosome-mediated aster formation.
Topics: Aurora Kinase B; Aurora Kinases; CSK Tyrosine-Protein Kinase; HeLa Cells; Humans; Mutation; Prometaphase; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-fyn; Spindle Apparatus; src-Family Kinases
PubMed: 22689581
DOI: 10.1074/jbc.M112.341578 -
Methods in Molecular Biology (Clifton,... 2017Understanding cell growth and cell division involves the study of regulatory events that occur in a cell cycle phase-dependent manner. Studies analyzing cell cycle...
Understanding cell growth and cell division involves the study of regulatory events that occur in a cell cycle phase-dependent manner. Studies analyzing cell cycle regulatory mechanisms and cell cycle progression invariably require synchronization of cell populations at specific cell cycle stages. Several methods have been established to synchronize cells, including serum deprivation, contact inhibition, centrifugal elutriation, and drug-dependent synchronization. Despite potential adverse cellular consequences of synchronizing cells by pharmacological agents, drug-dependent methods can be advantageous when studying later cell cycle events to ensure specific enrichment at selected mitotic stages. This chapter describes protocols used in our laboratory for isolating mitotic mammalian cells in a large-scale manner. In particular, we discuss the technical aspects of adherent or suspension cell isolation, the methods necessary to enrich cells at different mitotic stages and the optimized culture conditions.
Topics: Anaphase; Cell Culture Techniques; Cell Cycle; Fluorescent Antibody Technique; HeLa Cells; Humans; Metaphase; Mitosis; Prometaphase; Telophase
PubMed: 27815896
DOI: 10.1007/978-1-4939-6603-5_4 -
Oxidative Medicine and Cellular... 2017In zygotes, DNA damage delays the first cleavage to enable repair. Our previous study found that 0.03 mM hydrogen peroxide (HO) was the minimum concentration required...
In zygotes, DNA damage delays the first cleavage to enable repair. Our previous study found that 0.03 mM hydrogen peroxide (HO) was the minimum concentration required for induction of oxidative DNA damage in mouse zygotes and that this represented the most similar situation to the clinical phenomenon. In this study, we quantified the cleavage rates of cells in blastocysts at different developmental stages, followed by immunofluorescence to detect activation of -H2A histone family member X (a marker of DNA damage) in zygotes to confirm that oxidative DNA damage was induced in HO-treated zygotes. Monitoring H3S10P (phosphorylation of Ser10 on histone H3; a prometaphase/metaphase marker) levels at different hour postinsemination revealed that treatment of zygotes with 0.03 mM HO resulted in a prometaphase/metaphase delay. Furthermore, immunofluorescence staining for mitotic arrest deficient 2-like 1 and the protein kinase TTK, components of the spindle assembly checkpoint (SAC), suggested that this delay possibly involved SAC activation. These studies of the relationships between oxidative stress and SAC can promote the success rate of fertilization.
Topics: Animals; Blastocyst; Hydrogen Peroxide; Mad2 Proteins; Mice; Oxidative Stress; Spindle Apparatus; Zygote
PubMed: 29147457
DOI: 10.1155/2017/2103190 -
The EMBO Journal Jul 2009Regulation of BubR1 is central to the control of APC/C activity. We have found that BubR1 forms a complex with PCAF and is acetylated at lysine 250. Using mass...
Regulation of BubR1 is central to the control of APC/C activity. We have found that BubR1 forms a complex with PCAF and is acetylated at lysine 250. Using mass spectrometry and acetylated BubR1-specific antibodies, we have confirmed that BubR1 acetylation occurs at prometaphase. Importantly, BubR1 acetylation was required for checkpoint function, through the inhibition of ubiquitin-dependent BubR1 degradation. BubR1 degradation began before the onset of anaphase. It was noted that the pre-anaphase degradation was regulated by BubR1 acetylation. Degradation of an acetylation-mimetic form, BubR1-K250Q, was inhibited and chromosome segregation in cells expressing BubR1-K250Q was markedly delayed. By contrast, the acetylation-deficient mutant, BubR1-K250R, was unstable, and mitosis was accelerated in BubR1-K250R-expressing cells. Furthermore, we found that APC/C-Cdc20 was responsible for BubR1 degradation during mitosis. On the basis of our collective results, we propose that the acetylation status of BubR1 is a molecular switch that converts BubR1 from an inhibitor to a substrate of the APC/C complex, thus providing an efficient way to modulate APC/C activity and mitotic timing.
Topics: Acetylation; Amino Acid Sequence; Anaphase-Promoting Complex-Cyclosome; Animals; Cdc20 Proteins; Cell Cycle Proteins; Genes, cdc; HeLa Cells; Humans; Kinetochores; Mitosis; Molecular Sequence Data; Protein Serine-Threonine Kinases; Protein Stability; Sequence Alignment; Spindle Apparatus; Ubiquitin-Protein Ligase Complexes; Ubiquitination; p300-CBP Transcription Factors
PubMed: 19407811
DOI: 10.1038/emboj.2009.123 -
FEBS Letters May 2008The effect of double-strand DNA breaks (DSBs) on the spindle assembly checkpoint (SAC) has important implications with respect to the relationship between SAC function...
The effect of double-strand DNA breaks (DSBs) on the spindle assembly checkpoint (SAC) has important implications with respect to the relationship between SAC function and chromosome instability of cancer cells. Here, we demonstrate that induction of DSBs in mitosis results in prolonged hyper-phosphorylation of the SAC protein BubR1 and association of BubR1 with kinetochores in mammalian cells. Combining single cell time-lapse microscopy with immunofluorescence, flow cytometry, and Western blot analysis in synchronized cells, we provide evidence that DSBs activate BubR1, leading to prometaphase arrest. Accordingly, elimination of BubR1 expression by siRNA resulted in the abrogation of mitotic delay in response to chromosome damage. These results suggest that BubR1 links DNA damage to kinetochore-associated SAC function.
Topics: Chromosome Breakage; DNA Breaks, Double-Stranded; HeLa Cells; Humans; Prometaphase; Protein Serine-Threonine Kinases; Spindle Apparatus
PubMed: 18440313
DOI: 10.1016/j.febslet.2008.04.028