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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 -
Current Oncology (Toronto, Ont.) Nov 2022Kruppel-associated box (KRAB) proteins reportedly play a dual role in neoplastic transformation. At present, little is known about the function of the proteins encoded...
OBJECTIVE
Kruppel-associated box (KRAB) proteins reportedly play a dual role in neoplastic transformation. At present, little is known about the function of the proteins encoded by the human pogo transposable element derived with KRAB domain (POGK) gene. Herein, we evaluated the prognostic significance of POGK expression in patients with hepatocellular carcinoma (HCC).
METHODS
The data of HCC patients was downloaded from The Cancer Genome Atlas (TCGA) database. To determine the relationship between POGK and clinical features, logistic regression was applied. Cox regression and Kaplan-Meier analyses were used to evaluate the correlation between POGK and survival rates. Gene ontology (GO) analysis and Gene set enrichment analysis (GSEA) were conducted to identify the enriched pathways and functions associated with POGK.
RESULTS
A total of 374 HCC patients were identified in TCGA. POGK was significantly upregulated in HCC and correlated with tumor status ( = 0.036), race ( = 0.025), weight ( = 0.002), body mass index ( = 0.033), histologic grade ( < 0.001), and alpha-fetoprotein ( < 0.001). High POGK expression in HCC patients correlated with a poor outcome in terms of overall survival ( = 0.0018), progression-free survival ( = 0.0087), relapse-free survival ( = 0.045), and disease-specific survival ( = 0.014), according to Kaplan-Meier analysis. Receiver operating characteristic curve analysis showed that the area under the curve of POGK expression for HCC diagnosis was 0.891. GSEA showed that high POGK expression might activate mitotic prometaphase, kinesins, homologous DNA pairing and strand exchange, MET activates PTK2 signaling pathway, G1 to S cell cycle control, Aurora B pathway, ncRNAs involved in WNT signaling pathway, hepatitis C, and ncRNAs involved in the STAT3 signaling pathway. POGK expression correlated with the abundance of adaptive and innate immunocytes in HCC.
CONCLUSION
High expression of POGK has high diagnostic and prognostic values in patients with HCC. Moreover, POGK expression is correlated with immune infiltration in HCC.
Topics: Humans; Carcinoma, Hepatocellular; Liver Neoplasms; Gene Expression Regulation, Neoplastic; Biomarkers, Tumor; Neoplasm Recurrence, Local; Prognosis
PubMed: 36421335
DOI: 10.3390/curroncol29110682 -
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 -
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 -
Nano Letters Apr 2021Microtubules are highly strategic targets of cancer therapies. Small molecule antimitotic agents are so far the best chemotherapeutic medication in cancer treatment....
Microtubules are highly strategic targets of cancer therapies. Small molecule antimitotic agents are so far the best chemotherapeutic medication in cancer treatment. However, the high rate of neuropathy and drug resistance limit their clinical usage. Inspired by the multicomponent-targeting feature of molecular self-assembly (MSA) overcoming drug resistance, we synthesized peptide-based rotor molecules that self-assemble in response to the surrounding environment to target the microtubule array. The MSAs self-adjust morphologically in response to the pH change and viscosity variations during Golgi-endosome trafficking, escape trafficking cargos, and eventually bind to the microtubule array physically in a nonspecific manner. Such unrefined nano-bio interactions suppress regional tubulin polymerization triggering atypical prometaphase--metaphase oscillations to inhibit various cancer cells proliferating without inducing obvious neurotoxicity. The MSA also exerts potent antiproliferative effects in the subcutaneous cervix cancer xenograft tumor model equivalent to Cisplatin, better than the classic antimitotic drug Taxol.
Topics: Female; Humans; Metaphase; Microtubules; Neoplasms; Prometaphase; Tubulin
PubMed: 33756080
DOI: 10.1021/acs.nanolett.1c00233 -
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 -
Proceedings of the National Academy of... Jun 2020Topoisomerase IIα (TOP2A) is a core component of mitotic chromosomes and important for establishing mitotic chromosome condensation. The primary roles of TOP2A in...
Topoisomerase IIα (TOP2A) is a core component of mitotic chromosomes and important for establishing mitotic chromosome condensation. The primary roles of TOP2A in mitosis have been difficult to decipher due to its multiple functions across the cell cycle. To more precisely understand the role of TOP2A in mitosis, we used the auxin-inducible degron (AID) system to rapidly degrade the protein at different stages of the human cell cycle. Removal of TOP2A prior to mitosis does not affect prophase timing or the initiation of chromosome condensation. Instead, it prevents chromatin condensation in prometaphase, extends the length of prometaphase, and ultimately causes cells to exit mitosis without chromosome segregation occurring. Surprisingly, we find that removal of TOP2A from cells arrested in prometaphase or metaphase cause dramatic loss of compacted mitotic chromosome structure and conclude that TOP2A is crucial for maintenance of mitotic chromosomes. Treatments with drugs used to poison/inhibit TOP2A function, such as etoposide and ICRF-193, do not phenocopy the effects on chromosome structure of TOP2A degradation by AID. Our data point to a role for TOP2A as a structural chromosome maintenance enzyme locking in condensation states once sufficient compaction is achieved.
Topics: Chromosome Segregation; Chromosome Structures; Chromosomes, Human; Cytokinesis; DNA Topoisomerases, Type II; HCT116 Cells; Heterochromatin; Humans; Metaphase; Mitosis
PubMed: 32414923
DOI: 10.1073/pnas.2001760117 -
Scientific Reports Oct 2019We previously showed that curcumin, a phytopolyphenol found in turmeric (Curcuma longa), targets a series of enzymes in the ROS metabolic pathway, induces irreversible...
We previously showed that curcumin, a phytopolyphenol found in turmeric (Curcuma longa), targets a series of enzymes in the ROS metabolic pathway, induces irreversible growth arrest, and causes apoptosis. In this study, we tested Pentagamavunon-1 (PGV-1), a molecule related to curcumin, for its inhibitory activity on tumor cells in vitro and in vivo. PGV-1 exhibited 60 times lower GI compared to that of curcumin in K562 cells, and inhibited the proliferation of cell lines derived from leukemia, breast adenocarcinoma, cervical cancer, uterine cancer, and pancreatic cancer. The inhibition of growth by PGV-1 remained after its removal from the medium, which suggests that PGV-1 irreversibly prevents proliferation. PGV-1 specifically induced prometaphase arrest in the M phase of the cell cycle, and efficiently induced cell senescence and cell death by increasing intracellular ROS levels through inhibition of ROS-metabolic enzymes. In a xenograft mouse model, PGV-1 had marked anti-tumor activity with little side effects by oral administration, whereas curcumin rarely inhibited tumor formation by this administration. Therefore, PGV-1 is a potential therapeutic to induce tumor cell apoptosis with few side effects and low risk of relapse.
Topics: Administration, Oral; Alcohol Oxidoreductases; Animals; Antineoplastic Agents, Phytogenic; Carrier Proteins; Cell Death; Cell Division; Cell Movement; Cell Proliferation; Cellular Senescence; Curcumin; Gene Expression Regulation, Neoplastic; Glutathione S-Transferase pi; Glutathione Transferase; HEK293 Cells; HeLa Cells; Humans; K562 Cells; Lactoylglutathione Lyase; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; MCF-7 Cells; Mice, Nude; NAD(P)H Dehydrogenase (Quinone); Peroxiredoxins; Prometaphase; Reactive Oxygen Species; Tumor Burden; Xenograft Model Antitumor Assays
PubMed: 31619723
DOI: 10.1038/s41598-019-51244-3