bims-micesi 2022-03-06 papers

bims-micesi

Biomed News

on Mitotic cell signalling

Issue of 2022–03–06
seventeen papers selected by
Valentina Piano, Max Planck Institute of Molecular Physiology

The microtubule plus-end tracking protein Bik1 is required for chromosome congression.
PP2A-Cdc55 phosphatase regulates actomyosin ring contraction and septum formation during cytokinesis.
Evolutionary divergence of anaphase spindle mechanics in nematode embryos constrained by antagonistic pulling and viscous forces.
From the archives: Work on the mitotic spindle, root hair cell fate, and self-incompatibility highlights the wonderful world of the plant cell.
Chk1 dynamics in G2 phase upon replication stress predict daughter cell outcome.
Structures of SAS-6 coiled coil hold implications for the polarity of the centriolar cartwheel.
CDC7 kinase (DDK) inhibition disrupts DNA replication leading to mitotic catastrophe in Ewing sarcoma.
Meiosis-specific cohesin complexes display essential and distinct roles in mitotic embryonic stem cell chromosomes.
When More is Less: Heritable Gain-of-Function Chk1 Mutations Impair Human Fertility.
Wnt signaling polarizes cortical actin polymerization to increase daughter cell asymmetry.
The control of transcriptional memory by stable mitotic bookmarking.
Sister chromatids separate during anaphase in a three-stage program as directed by interaxis bridges.
MiNuGAN: Dual Segmentation of Mitoses and Nuclei Using Conditional GANs on Multi-center Breast H&E Images.
Spindle cell/sclerosing rhabdomyosarcoma with DCTN1::ALK fusion: broadening the molecular spectrum with potential therapeutic implications.
Cdc48Ufd1/Npl4 segregase removes mislocalized centromeric histone H3 variant CENP-A from non-centromeric chromatin.
Probing the Role of Aurora Kinase A Threonylation with Site-Specific Lysine Threonylation.
PLK1 inhibition selectively induces apoptosis in ARID1A deficient cells through uncoupling of oxygen consumption from ATP production.

Mol Biol Cell. 2022 Mar 02. mbcE21100500

The microtubule plus-end tracking protein Bik1 is required for chromosome congression.

Alexander Julner, Marjan Abbasi, Victoria Menéndez-Benito.

During mitosis, sister chromatids congress on both sides of the spindle equator to facilitate the correct partitioning of the genomic material. Chromosome congression requires a finely tuned control of microtubule dynamics by the kinesin motor proteins. In Saccharomyces cerevisiae, the kinesin proteins Cin8, Kip1, and Kip3 have a pivotal role in chromosome congression. It has been hypothesized that additional proteins that modulate microtubule dynamics are also involved. Here, we show that the microtubule plus-end tracking protein Bik1 - the budding yeast ortholog of CLIP-170 - is essential for chromosome congression. We find that nuclear Bik1 localizes to the kinetochores in a cell-cycle-dependent manner. Disrupting the nuclear pool of Bik1 with a nuclear export signal (Bik1-NES) leads to a slower cell cycle progression characterized by a delayed metaphase-anaphase transition. Bik1-NES cells have mispositioned kinetochores along the spindle in metaphase. Furthermore, using proximity-dependent methods, we identify Cin8 as an interaction partner of Bik1. Deleting CIN8 reduces the amount of Bik1 at the spindle. In contrast, Cin8 retains its typical bilobed distribution in the Bik1-NES mutant and does not localize to the unclustered kinetochores. Thus, we propose that Bik1 functions with Cin8 to regulate kinetochore-microtubule dynamics for correct kinetochore positioning and chromosome congression.

DOI: https://doi.org/10.1091/mbc.E21-10-0500
Cell Mol Life Sci. 2022 Mar 01. 79(3): 165

PP2A-Cdc55 phosphatase regulates actomyosin ring contraction and septum formation during cytokinesis.

Yolanda Moyano-Rodríguez, David Vaquero, Odena Vilalta-Castany, Magdalena Foltman, Alberto Sanchez-Diaz, Ethel Queralt.

  Eukaryotic cells divide and separate all their components after chromosome segregation by a process called cytokinesis to complete cell division. Cytokinesis is highly regulated by the recruitment of the components to the division site and through post-translational modifications such as phosphorylations. The budding yeast mitotic kinases Cdc28-Clb2, Cdc5, and Dbf2-Mob1 phosphorylate several cytokinetic proteins contributing to the regulation of cytokinesis. The PP2A-Cdc55 phosphatase regulates mitosis counteracting Cdk1- and Cdc5-dependent phosphorylation. This prompted us to propose that PP2A-Cdc55 could also be counteracting the mitotic kinases during cytokinesis. Here we show that in the absence of Cdc55, AMR contraction and the primary septum formation occur asymmetrically to one side of the bud neck supporting a role for PP2A-Cdc55 in cytokinesis regulation. In addition, by in vivo and in vitro assays, we show that PP2A-Cdc55 dephosphorylates the chitin synthase II (Chs2 in budding yeast) a component of the Ingression Progression Complexes (IPCs) involved in cytokinesis. Interestingly, the non-phosphorylable version of Chs2 rescues the asymmetric AMR contraction and the defective septa formation observed in cdc55∆ mutant cells. Therefore, timely dephosphorylation of the Chs2 by PP2A-Cdc55 is crucial for proper actomyosin ring contraction. These findings reveal a new mechanism of cytokinesis regulation by the PP2A-Cdc55 phosphatase and extend our knowledge of the involvement of multiple phosphatases during cytokinesis.

Keywords:  AMR contraction; Cell Cycle; Cyk3; Cytokinesis; Hof1; Ingression progression complexes (IPCs); Myo1; PP2A-Cdc55

DOI:  https://doi.org/10.1007/s00018-022-04209-1
Mol Biol Cell. 2022 Mar 02. mbcE21100532

Evolutionary divergence of anaphase spindle mechanics in nematode embryos constrained by antagonistic pulling and viscous forces.

Dhruv Khatri, Thibault Brugière, Chaitanya A Athale, Marie Delattre.

Cellular functions like cell division are remarkably conserved across phyla. However the evolutionary principles of cellular organization that drive it are less well explored. Thus, an essential question remains: to what extent cellular parameters evolve without altering the basic function they sustain? Here we have observed 6 different nematode species for which the mitotic spindle is positioned asymmetrically during the first embryonic division. Whereas the C. elegans spindle undergoes oscillations during its displacement, the spindle elongates without oscillations in other species. We asked which evolutionary changes in biophysical parameters could explain differences in spindle motion while maintaining a constant output. Using laser microsurgery of the spindle we revealed that all species are subjected to cortical pulling forces, of varying magnitudes. Using a viscoelastic model to fit the recoil trajectories and with an independent measurement of cytoplasmic viscosity, we extracted the values of cytoplasmic drag, cortical pulling forces and spindle elasticity for all species. We found large variations in cytoplasmic viscosity whereas cortical pulling forces and elasticity were often more constrained. In agreement with previous simulations, we found that increased viscosity correlates with decreased oscillation speeds across species. However, the absence of oscillations despite low viscosity in some species, can only be explained by smaller pulling forces. Consequently, we find that spindle mobility across the species analyzed here is characterized by a tradeoff between cytoplasmic viscosity and pulling forces normalized by the size of the embryo. Our work provides a framework for understanding mechanical constraints on evolutionary diversification of spindle mobility.

DOI: https://doi.org/10.1091/mbc.E21-10-0532
Plant Cell. 2022 Jan 20. 34(1): 495-496

From the archives: Work on the mitotic spindle, root hair cell fate, and self-incompatibility highlights the wonderful world of the plant cell.

Nancy A Eckardt.

DOI: https://doi.org/10.1093/plcell/koab274
Dev Cell. 2022 Feb 23. pii: S1534-5807(22)00079-X. [Epub ahead of print]

Chk1 dynamics in G2 phase upon replication stress predict daughter cell outcome.

Vicente Lebrec, Marion Poteau, Jean-Philippe Morretton, Olivier Gavet.

  In human cells, ATR/Chk1 signaling couples S phase exit with the expression of mitotic inducers and prevents premature mitosis upon replication stress (RS). Nonetheless, under-replicated DNA can persist at mitosis, prompting chromosomal instability. To decipher how the DNA replication checkpoint (DRC) allows cells to enter mitosis over time upon RS, we developed a FRET-based Chk1 activity sensor. During unperturbed growth, a basal Chk1 activity level is sustained throughout S phase and relies on replication origin firing. Incremental RS triggers stepwise Chk1 over-activation that delays S-phase, suggesting a rheostat-like role for DRC coupled with the replication machinery. Upon RS, Chk1 is inactivated as DNA replication terminates but surprisingly is reactivated in a subset of G2 cells, which relies on Cdk1/2 and Plk1 and prevents mitotic entry. Cells can override active Chk1 signaling and reach mitosis onset, revealing checkpoint adaptation. Cell division following Chk1 reactivation in G2 results in a p53/p21-dependent G1 arrest, eliminating the daughter cells from proliferation.

Keywords:  DNA replication checkpoint; FRET biosensor; G2 phase; cell cycle recovery; checkpoint adaptation; checkpoint kinase 1; replication stress

DOI:  https://doi.org/10.1016/j.devcel.2022.02.013
Structure. 2022 Feb 22. pii: S0969-2126(22)00041-7. [Epub ahead of print]

Structures of SAS-6 coiled coil hold implications for the polarity of the centriolar cartwheel.

Anastassia L Kantsadi, Georgios N Hatzopoulos, Pierre Gönczy, Ioannis Vakonakis.

  Centrioles are eukaryotic organelles that template the formation of cilia and flagella, as well as organize the microtubule network and the mitotic spindle in animal cells. Centrioles have proximal-distal polarity and a 9-fold radial symmetry imparted by a likewise symmetrical central scaffold, the cartwheel. The spindle assembly abnormal protein 6 (SAS-6) self-assembles into 9-fold radially symmetric ring-shaped oligomers that stack via an unknown mechanism to form the cartwheel. Here, we uncover a homo-oligomerization interaction mediated by the coiled-coil domain of SAS-6. Crystallographic structures of Chlamydomonas reinhardtii SAS-6 coiled-coil complexes suggest this interaction is asymmetric, thereby imparting polarity to the cartwheel. Using a cryoelectron microscopy (cryo-EM) reconstitution assay, we demonstrate that amino acid substitutions disrupting this asymmetric association also impair SAS-6 ring stacking. Our work raises the possibility that the asymmetric interaction inherent to SAS-6 coiled-coil provides a polar element for cartwheel assembly, which may assist the establishment of the centriolar proximal-distal axis.

Keywords:  SAS-6; asymmetry; cartwheel; centriole; coiled-coil; complex; crystallography; electron microscopy; self-assembly; stacking

DOI:  https://doi.org/10.1016/j.str.2022.02.005
Cell Death Discov. 2022 Feb 26. 8(1): 85

CDC7 kinase (DDK) inhibition disrupts DNA replication leading to mitotic catastrophe in Ewing sarcoma.

Jeffrey C Martin, Jennie R Sims, Ajay Gupta, Tamara J Hagoel, Lingqiu Gao, Miranda L Lynch, Anna Woloszynska, Thomas Melendy, Jeremy F Kane, Joseph Kuechle, Joyce E Ohm.

Ewing sarcoma is the second most common bone malignancy in children and adolescents. In recent years, a large body of evidence has emerged that suggests Ewing tumors harbor large amounts of replication stress (RS). CDC7, also known as DDK (DBF4-dependent kinase), is a serine/threonine kinase that is involved in a diverse array of cellular functions including the regulation of DNA replication initiation and activation of the RS response. Due to DDK's diverse roles during replication, coupled with the fact that there is an increased level of RS within Ewing tumors, we hypothesized that Ewing sarcoma cells would be particularly vulnerable to DDK inhibition. Here, we report that DDK inhibition resulted a significant reduction in cell viability and the induction of apoptosis, specifically in Ewing sarcoma cells. Treatment with DDK inhibitors dramatically reduced the rate of replication, prolonged S-phase, and led to a pronounced increase in phospho-CDC2 (Y15), indicating delay of mitotic entry. The induction of cell death corresponded to mitotic exit and G1 entry, suggesting improper mitotic progression. In accordance with this, we find that DDK inhibition caused premature mitotic entry resulting in mitotic abnormalities such as anaphase bridges, lagging chromosomes, and cells with >2 poles in Ewing sarcoma cells. This abnormal progression through mitosis resulted in mitotic catastrophe as evidenced by the formation of micronuclei and induction of DNA damage. Together, these findings suggest that DDK activity is required for the faithful and timely completion of DNA replication in Ewing cells and that DDK inhibition may present a viable therapeutic strategy for the treatment of Ewing sarcoma.

DOI: https://doi.org/10.1038/s41420-022-00877-x
Genome Biol. 2022 Mar 03. 23(1): 70

Meiosis-specific cohesin complexes display essential and distinct roles in mitotic embryonic stem cell chromosomes.

Eui-Hwan Choi, Seobin Yoon, Young Eun Koh, Tae Kyung Hong, Jeong Tae Do, Bum-Kyu Lee, Yoonsoo Hahn, Keun P Kim.

   BACKGROUND: Cohesin is a chromosome-associated SMC-kleisin complex that mediates sister chromatid cohesion, recombination, and most chromosomal processes during mitosis and meiosis. However, it remains unclear whether meiosis-specific cohesin complexes are functionally active in mitotic chromosomes.
RESULTS: Through high-resolution 3D-structured illumination microscopy (3D-SIM) and functional analyses, we report multiple biological processes associated with the meiosis-specific cohesin components, α-kleisin REC8 and STAG3, and the distinct loss of function of meiotic cohesin during the cell cycle of embryonic stem cells (ESCs). First, we show that STAG3 is required for the efficient localization of REC8 to the nucleus by interacting with REC8. REC8-STAG3-containing cohesin regulates topological properties of chromosomes and maintains sister chromatid cohesion. Second, REC8-cohesin has additional sister chromatid cohesion roles in concert with mitotic RAD21-cohesin on ESC chromosomes. SIM imaging of REC8 and RAD21 co-staining revealed that the two types of α-kleisin subunits exhibited distinct loading patterns along ESC chromosomes. Third, knockdown of REC8 or RAD21-cohesin not only leads to higher rates of premature sister chromatid separation and delayed replication fork progression, which can cause proliferation and developmental defects, but also enhances chromosome compaction by hyperloading of retinoblastoma protein-condensin complexes from the prophase onward.
CONCLUSIONS: Our findings indicate that the delicate balance between mitotic and meiotic cohesins may regulate ESC-specific chromosomal organization and the mitotic program.

Keywords:  Chromosome; Cohesin; Embryonic stem cell; Meiosis; Mitosis

DOI:  https://doi.org/10.1186/s13059-022-02632-y
FEBS J. 2022 Mar 01.

When More is Less: Heritable Gain-of-Function Chk1 Mutations Impair Human Fertility.

David A Gillespie.

  Heritable loss-of-function mutations in genes encoding key regulators of DNA repair and genome stability can result in degenerative progeroid and/ or cancer predisposition syndromes, however such mutations have never been found to affect the Chk1 protein kinase, despite its central role in DNA damage signalling and checkpoint activation. Remarkably, two recent reports now demonstrate that heritable, gain-of-function mutations within the Chk1 C-terminal regulatory domain can cause female infertility in humans. In vitro, oocytes from individuals heterozygous for such mutant Chk1 alleles fail to undergo the first mitotic division after fertilization owing to arrest in G2 phase of the cell cycle. This arrest results from inhibition of the master regulator of mitosis, the cyclin-dependent kinase CDK1, through the same molecular mechanisms that are engaged by activated Chk1 to impose G2 checkpoint arrest in somatic cells bearing DNA damage. Remarkably, the failure of this first zygotic division in heterozygotes in vitro can be rescued through treatment with selective Chk1 inhibitor drugs, allowing development of apparently normal blastocysts and offering hope that a pharmacological solution to this cause of infertility may be possible.

Keywords:  Chk1; DNA damage; cell cycle; human fertility; protein kinase inhibitor

DOI:  https://doi.org/10.1111/febs.16415
Cell Discov. 2022 Mar 01. 8(1): 22

Wnt signaling polarizes cortical actin polymerization to increase daughter cell asymmetry.

Yongping Chai, Dong Tian, Zhiwen Zhu, Yuxiang Jiang, Shanjin Huang, Dou Wu, Guangshuo Ou, Wei Li.

Asymmetric positioning of the mitotic spindle contributes to the generation of two daughter cells with distinct sizes and fates. Here, we investigated an asymmetric division in the Caenorhabditis elegans Q neuroblast lineage. In this division, beginning with an asymmetrically positioned spindle, the daughter-cell size differences continuously increased during cytokinesis, and the smaller daughter cell in the posterior eventually underwent apoptosis. We found that Arp2/3-dependent F-actin assembled in the anterior but not posterior cortex during division, suggesting that asymmetric expansion forces generated by actin polymerization may enlarge the anterior daughter cell. Consistent with this, inhibition of cortical actin polymerization or artificially equalizing actin assembly led to symmetric cell division. Furthermore, disruption of the Wnt gradient or its downstream components impaired asymmetric cortical actin assembly and caused symmetric division. Our results show that Wnt signaling establishes daughter cell asymmetry by polarizing cortical actin polymerization in a dividing cell.

DOI: https://doi.org/10.1038/s41421-022-00376-4
Nat Commun. 2022 Mar 04. 13(1): 1176

The control of transcriptional memory by stable mitotic bookmarking.

Maëlle Bellec, Jérémy Dufourt, George Hunt, Hélène Lenden-Hasse, Antonio Trullo, Amal Zine El Aabidine, Marie Lamarque, Marissa M Gaskill, Heloïse Faure-Gautron, Mattias Mannervik, Melissa M Harrison, Jean-Christophe Andrau, Cyril Favard, Ovidiu Radulescu, Mounia Lagha.

To maintain cellular identities during development, gene expression profiles must be faithfully propagated through cell generations. The reestablishment of gene expression patterns upon mitotic exit is mediated, in part, by transcription factors (TF) mitotic bookmarking. However, the mechanisms and functions of TF mitotic bookmarking during early embryogenesis remain poorly understood. In this study, taking advantage of the naturally synchronized mitoses of Drosophila early embryos, we provide evidence that GAGA pioneer factor (GAF) acts as a stable mitotic bookmarker during zygotic genome activation. We show that, during mitosis, GAF remains associated to a large fraction of its interphase targets, including at cis-regulatory sequences of key developmental genes with both active and repressive chromatin signatures. GAF mitotic targets are globally accessible during mitosis and are bookmarked via histone acetylation (H4K8ac). By monitoring the kinetics of transcriptional activation in living embryos, we report that GAF binding establishes competence for rapid activation upon mitotic exit.

DOI: https://doi.org/10.1038/s41467-022-28855-y
Proc Natl Acad Sci U S A. 2022 Mar 08. 119(10): e2123363119

Sister chromatids separate during anaphase in a three-stage program as directed by interaxis bridges.

Lingluo Chu, Zheng Zhang, Maria Mukhina, Denise Zickler, Nancy Kleckner.

  Significance A central feature of mitosis is segregation of sister chromatids to opposite poles during anaphase. Our recent work revealed that sister chromatids are linked by robust structural bridges built on topological sister/sister catenations. This unexpected finding implies that separation of sister chromatids is more complex than previously thought. The present study reveals that bridges are removed in a highly programmed three-stage process, all licensed by anaphase onset and cohesin removal, and all promoted by distinct types of intersister separation forces. Removal of bridge-associated cohesin and topoisomerase II-mediated decatenation plays a central role. These findings raise the possibility that the presence and programmed removal of bridges are required for smooth, synchronous, and regular movement of sisters to opposite poles.

Keywords:  TopoII decatenation; anaphase; cohesin; interaxis bridges; mitosis

DOI:  https://doi.org/10.1073/pnas.2123363119
J Pathol Inform. 2022 ;13 100002

MiNuGAN: Dual Segmentation of Mitoses and Nuclei Using Conditional GANs on Multi-center Breast H&E Images.

Salar Razavi, Fariba D Khameneh, Hana Nouri, Dimitrios Androutsos, Susan J Done, April Khademi.

  Breast cancer is the second most commonly diagnosed type of cancer among women as of 2021. Grading of histopathological images is used to guide breast cancer treatment decisions and a critical component of this is a mitotic score, which is related to tumor aggressiveness. Manual mitosis counting is an extremely tedious manual task, but automated approaches can be used to overcome inefficiency and subjectivity. In this paper, we propose an automatic mitosis and nuclear segmentation method for a diverse set of H&E breast cancer pathology images. The method is based on a conditional generative adversarial network to segment both mitoses and nuclei at the same time. Architecture optimizations are investigated, including hyper parameters and the addition of a focal loss. The accuracy of the proposed method is investigated using images from multiple centers and scanners, including TUPAC16, ICPR14 and ICPR12 datasets. In TUPAC16, we use 618 carefully annotated images of size 256×256 scanned at 40×. TUPAC16 is used to train the model, and segmentation performance is measured on the test set for both nuclei and mitoses. Results on 200 held-out testing images from the TUPAC16 dataset were mean DSC = 0.784 and 0.721 for nuclear and mitosis, respectively. On 202 ICPR12 images, mitosis segmentation accuracy had a mean DSC = 0.782, indicating the model generalizes well to unseen datasets. For datasets that had mitosis centroid annotations, which included 200 TUPAC16, 202 ICPR12 and 524 ICPR14, a mean F1-score of 0.854 was found indicating high mitosis detection accuracy.

Keywords:  Breast cancer; Computer-aided detection; Focal loss; Generative adversarial network; Mitosis detection; Semantic segmentation

DOI:  https://doi.org/10.1016/j.jpi.2022.100002
Virchows Arch. 2022 Mar 01.

Spindle cell/sclerosing rhabdomyosarcoma with DCTN1::ALK fusion: broadening the molecular spectrum with potential therapeutic implications.

C K Fung, Chit Chow, W K Chan, Eric W K Choi, K F To, John K C Chan, Wah Cheuk.

  Spindle cell/sclerosing rbabdomyosarcoma (RMS) is a recently characterized variant of RMS with several distinct molecular subtypes. We describe an example occurring in the tongue of a 10-year-old boy with a novel DCTN1::ALK fusion. The tumor exhibited infiltrative growth and was comprised of fascicles and focally whorls of spindle cells with eosinophilic cytoplasm, in a collagenous or myxoid stroma. Moderate cytologic atypia, mitotic activity (2/10 HPFs), and perineural invasion were identified. The tumor cells expressed actin, desmin, MyoD1, myogenin, and ALK. An in-frame fusion between DCTN1 exon 26 and ALK exon 20 was detected by RNA sequencing, which was confirmed by split reads and supported by FISH studies. The tumor showed an indolent behavior with local recurrence 3 years after excision. This study broadens the molecular spectrum of spindle cell/sclerosing RMS and this molecular aberration may represent a potential therapeutic target for unresectable or disseminated disease.

Keywords:  DCTN1 ALK; RNA seq; Spindle cell/sclerosing rhabdomyosarcoma

DOI:  https://doi.org/10.1007/s00428-022-03305-8
Nucleic Acids Res. 2022 Mar 02. pii: gkac135. [Epub ahead of print]

Cdc48Ufd1/Npl4 segregase removes mislocalized centromeric histone H3 variant CENP-A from non-centromeric chromatin.

Kentaro Ohkuni, Loran Gliford, Wei-Chun Au, Evelyn Suva, Peter Kaiser, Munira A Basrai.

Restricting the localization of CENP-A (Cse4 in Saccharomyces cerevisiae) to centromeres prevents chromosomal instability (CIN). Mislocalization of overexpressed CENP-A to non-centromeric chromatin contributes to CIN in budding and fission yeasts, flies, and humans. Overexpression and mislocalization of CENP-A is observed in cancers and is associated with increased invasiveness. Mechanisms that remove mislocalized CENP-A and target it for degradation have not been defined. Here, we report that Cdc48 and its cofactors Ufd1 and Npl4 facilitate the removal of mislocalized Cse4 from non-centromeric chromatin. Defects in removal of mislocalized Cse4 contribute to lethality of overexpressed Cse4 in cdc48,ufd1 andnpl4 mutants. High levels of polyubiquitinated Cse4 and mislocalization of Cse4 are observed in cdc48-3, ufd1-2 and npl4-1mutants even under normal physiological conditions, thereby defining polyubiquitinated Cse4 as the substrate of the ubiquitin directed segregase Cdc48Ufd1/Npl4. Accordingly, Npl4, the ubiquitin binding receptor, associates with mislocalized Cse4, and this interaction is dependent on Psh1-mediated polyubiquitination of Cse4. In summary, we provide the first evidence for a mechanism that facilitates the removal of polyubiquitinated and mislocalized Cse4 from non-centromeric chromatin. Given the conservation of Cdc48Ufd1/Npl4 in humans, it is likely that defects in such pathways may contribute to CIN in human cancers.

DOI: https://doi.org/10.1093/nar/gkac135
ACS Chem Biol. 2022 Mar 01.

Probing the Role of Aurora Kinase A Threonylation with Site-Specific Lysine Threonylation.

Jia Zang, Yulin Chen, Chao Liu, Shixian Lin.

Protein post-translational modifications play central roles in regulating protein functions. Lysine threonylation is a newly discovered reversible post-translational modification. However, the biological effect of lysine threonylation on proteins remains largely elusive. Here we report a chemical biology approach for site-specific incorporation of Nε-threonyllysine into proteins with high efficiency and investigate the biological effect of lysine threonylation on Aurora kinase A. Using this unnatural amino acid mutagenesis approach, we find that threonylation of Lys162 of Aurora kinase A inhibits its kinase activity both in vitro and in vivo and that the inhibitory effect can be reversed by the deacetylase Sirtuin 3, which removes the threonylated group from the lysine. Additionally, threonylation of Aurora kinase A makes its substrate p53 more stable in the cell. Therefore, our study demonstrates that site-specific lysine threonylation is a powerful method for probing the biological effect of protein threonylation.

DOI: https://doi.org/10.1021/acschembio.1c00682
Oncogene. 2022 Mar 02.

PLK1 inhibition selectively induces apoptosis in ARID1A deficient cells through uncoupling of oxygen consumption from ATP production.

Upadhyayula S Srinivas, Norbert S C Tay, Patrick Jaynes, Akshaya Anbuselvan, Gokula K Ramachandran, Joanna D Wardyn, Michal M Hoppe, Phuong Mai Hoang, Yanfen Peng, Sherlly Lim, May Yin Lee, Praveen C Peethala, Omer An, Akshay Shendre, Bryce W Q Tan, Sherlyn Jemimah, Manikandan Lakshmanan, Longyu Hu, Rekha Jakhar, Karishma Sachaphibulkij, Lina H K Lim, Shazib Pervaiz, Karen Crasta, Henry Yang, Patrick Tan, Chao Liang, Lena Ho, Vartika Khanchandani, Dennis Kappei, Wei Peng Yong, David S P Tan, Matteo Bordi, Silvia Campello, Wai Leong Tam, Christian Frezza, Anand D Jeyasekharan.

Inhibitors of the mitotic kinase PLK1 yield objective responses in a subset of refractory cancers. However, PLK1 overexpression in cancer does not correlate with drug sensitivity, and the clinical development of PLK1 inhibitors has been hampered by the lack of patient selection marker. Using a high-throughput chemical screen, we discovered that cells deficient for the tumor suppressor ARID1A are highly sensitive to PLK1 inhibition. Interestingly this sensitivity was unrelated to canonical functions of PLK1 in mediating G2/M cell cycle transition. Instead, a whole-genome CRISPR screen revealed PLK1 inhibitor sensitivity in ARID1A deficient cells to be dependent on the mitochondrial translation machinery. We find that ARID1A knock-out (KO) cells have an unusual mitochondrial phenotype with aberrant biogenesis, increased oxygen consumption/expression of oxidative phosphorylation genes, but without increased ATP production. Using expansion microscopy and biochemical fractionation, we see that a subset of PLK1 localizes to the mitochondria in interphase cells. Inhibition of PLK1 in ARID1A KO cells further uncouples oxygen consumption from ATP production, with subsequent membrane depolarization and apoptosis. Knockdown of specific subunits of the mitochondrial ribosome reverses PLK1-inhibitor induced apoptosis in ARID1A deficient cells, confirming specificity of the phenotype. Together, these findings highlight a novel interphase role for PLK1 in maintaining mitochondrial fitness under metabolic stress, and a strategy for therapeutic use of PLK1 inhibitors. To translate these findings, we describe a quantitative microscopy assay for assessment of ARID1A protein loss, which could offer a novel patient selection strategy for the clinical development of PLK1 inhibitors in cancer.

DOI: https://doi.org/10.1038/s41388-022-02219-8