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1.
Nat Commun ; 12(1): 4917, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389714

RESUMO

APOBEC3A is a cytidine deaminase driving mutagenesis in tumors. While APOBEC3A-induced mutations are common, APOBEC3A expression is rarely detected in cancer cells. This discrepancy suggests a tightly controlled process to regulate episodic APOBEC3A expression in tumors. In this study, we find that both viral infection and genotoxic stress transiently up-regulate APOBEC3A and pro-inflammatory genes using two distinct mechanisms. First, we demonstrate that STAT2 promotes APOBEC3A expression in response to foreign nucleic acid via a RIG-I, MAVS, IRF3, and IFN-mediated signaling pathway. Second, we show that DNA damage and DNA replication stress trigger a NF-κB (p65/IkBα)-dependent response to induce expression of APOBEC3A and other innate immune genes, independently of DNA or RNA sensing pattern recognition receptors and the IFN-signaling response. These results not only reveal the mechanisms by which tumors could episodically up-regulate APOBEC3A but also highlight an alternative route to stimulate the immune response after DNA damage independently of cGAS/STING or RIG-I/MAVS.


Assuntos
Citidina Desaminase/genética , Dano ao DNA , Regulação da Expressão Gênica , Imunidade/genética , Proteínas/genética , Transdução de Sinais/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Citidina Desaminase/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células THP-1 , Fator de Transcrição RelA/metabolismo , Regulação para Cima , Vírus/crescimento & desenvolvimento
2.
Mol Cell ; 81(4): 739-755.e7, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33606975

RESUMO

Cyclic GMP-AMP synthase (cGAS) recognition of cytosolic DNA is critical for the immune response to cancer and pathogen infection. Here, we discover that cGAS-DNA phase separation is required to resist negative regulation and allow efficient sensing of immunostimulatory DNA. We map the molecular determinants of cGAS condensate formation and demonstrate that phase separation functions to limit activity of the cytosolic exonuclease TREX1. Mechanistically, phase separation forms a selective environment that suppresses TREX1 catalytic function and restricts DNA degradation to an outer shell at the droplet periphery. We identify a TREX1 mutation associated with the severe autoimmune disease Aicardi-Goutières syndrome that increases penetration of TREX1 into the repressive droplet interior and specifically impairs degradation of phase-separated DNA. Our results define a critical function of cGAS-DNA phase separation and reveal a molecular mechanism that balances cytosolic DNA degradation and innate immune activation.


Assuntos
Doenças Autoimunes do Sistema Nervoso/enzimologia , Citosol/metabolismo , DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Mutação , Malformações do Sistema Nervoso/enzimologia , Nucleotidiltransferases/metabolismo , Fosfoproteínas/metabolismo , Doenças Autoimunes do Sistema Nervoso/genética , Catálise , Linhagem Celular Tumoral , DNA/genética , Exodesoxirribonucleases/genética , Células HEK293 , Humanos , Malformações do Sistema Nervoso/genética , Nucleotidiltransferases/genética , Fosfoproteínas/genética
3.
Mol Cell ; 81(4): 724-738.e9, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33476576

RESUMO

Micronuclei are aberrant nuclear compartments that can form as a result of chromosome mis-segregation. Frequent loss of micronuclear envelope integrity exposes DNA to the cytoplasm, leading to chromosome fragmentation and immune activation. Here, we use micronuclei purification to show that the endoplasmic reticulum (ER)-associated nuclease TREX1 inhibits cGAS activation at micronuclei by degrading micronuclear DNA upon micronuclear envelope rupture. We demonstrate that the ER accesses ruptured micronuclei and plays a critical role in enabling TREX1 nucleolytic attack. TREX1 mutations, previously implicated in immune disease, untether TREX1 from the ER, disrupt TREX1 localization to micronuclei, diminish micronuclear DNA damage, and enhance cGAS activation. These results establish ER-directed resection of micronuclear DNA by TREX1 as a critical regulator of cytosolic DNA sensing in chromosomally unstable cells and provide a mechanistic basis for the importance of TREX1 ER tethering in preventing autoimmunity.


Assuntos
Dano ao DNA , Retículo Endoplasmático/metabolismo , Exodesoxirribonucleases/metabolismo , Micronúcleos com Defeito Cromossômico , Mutação , Nucleotidiltransferases/metabolismo , Fosfoproteínas/metabolismo , Retículo Endoplasmático/genética , Ativação Enzimática/genética , Exodesoxirribonucleases/genética , Células HEK293 , Humanos , Nucleotidiltransferases/genética , Fosfoproteínas/genética , Transporte Proteico/genética
4.
Cell ; 183(1): 197-210.e32, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33007263

RESUMO

Cancer genomes often harbor hundreds of somatic DNA rearrangement junctions, many of which cannot be easily classified into simple (e.g., deletion) or complex (e.g., chromothripsis) structural variant classes. Applying a novel genome graph computational paradigm to analyze the topology of junction copy number (JCN) across 2,778 tumor whole-genome sequences, we uncovered three novel complex rearrangement phenomena: pyrgo, rigma, and tyfonas. Pyrgo are "towers" of low-JCN duplications associated with early-replicating regions, superenhancers, and breast or ovarian cancers. Rigma comprise "chasms" of low-JCN deletions enriched in late-replicating fragile sites and gastrointestinal carcinomas. Tyfonas are "typhoons" of high-JCN junctions and fold-back inversions associated with expressed protein-coding fusions, breakend hypermutation, and acral, but not cutaneous, melanomas. Clustering of tumors according to genome graph-derived features identified subgroups associated with DNA repair defects and poor prognosis.


Assuntos
Variação Estrutural do Genoma/genética , Genômica/métodos , Neoplasias/genética , Inversão Cromossômica/genética , Cromotripsia , Variações do Número de Cópias de DNA/genética , Rearranjo Gênico/genética , Genoma Humano/genética , Humanos , Mutação/genética , Sequenciamento Completo do Genoma/métodos
5.
DNA Repair (Amst) ; 94: 102905, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32818816

RESUMO

The APOBEC family of cytidine deaminases has been proposed to represent a major enzymatic source of mutations in cancer. Here, we summarize available evidence that links APOBEC deaminases to cancer mutagenesis. We also highlight newly identified human cell models of APOBEC mutagenesis, including cancer cell lines with suspected endogenous APOBEC activity and a cell system of telomere crisis-associated mutations. Finally, we draw on recent data to propose potential causes of APOBEC misregulation in cancer, including the instigating factors, the relevant mutator(s), and the mechanisms underlying generation of the genome-dispersed and clustered APOBEC-induced mutations.


Assuntos
Desaminases APOBEC/genética , Mutação , Neoplasias/enzimologia , Animais , Humanos , Mutagênese , Neoplasias/genética
6.
Nat Genet ; 52(9): 884-890, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32719516

RESUMO

Chromothripsis and kataegis are frequently observed in cancer and may arise from telomere crisis, a period of genome instability during tumorigenesis when depletion of the telomere reserve generates unstable dicentric chromosomes1-5. Here we examine the mechanism underlying chromothripsis and kataegis by using an in vitro telomere crisis model. We show that the cytoplasmic exonuclease TREX1, which promotes the resolution of dicentric chromosomes4, plays a prominent role in chromothriptic fragmentation. In the absence of TREX1, the genome alterations induced by telomere crisis primarily involve breakage-fusion-bridge cycles and simple genome rearrangements rather than chromothripsis. Furthermore, we show that the kataegis observed at chromothriptic breakpoints is the consequence of cytosine deamination by APOBEC3B. These data reveal that chromothripsis and kataegis arise from a combination of nucleolytic processing by TREX1 and cytosine editing by APOBEC3B.


Assuntos
Citidina Desaminase/genética , Exodesoxirribonucleases/genética , Fosfoproteínas/genética , Telômero/genética , Linhagem Celular Tumoral , Cromotripsia , Citosina Desaminase/genética , Instabilidade Genômica/genética , Humanos , Mutação/genética , Neoplasias/genética , Células U937
7.
Annu Rev Cell Dev Biol ; 36: 85-114, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32692592

RESUMO

The nuclear envelope is often depicted as a static barrier that regulates access between the nucleus and the cytosol. However, recent research has identified many conditions in cultured cells and in vivo in which nuclear membrane ruptures cause the loss of nuclear compartmentalization. These conditions include some that are commonly associated with human disease, such as migration of cancer cells through small spaces and expression of nuclear lamin disease mutations in both cultured cells and tissues undergoing nuclear migration. Nuclear membrane ruptures are rapidly repaired in the nucleus but persist in nuclear compartments that form around missegregated chromosomes called micronuclei. This review summarizes what is known about the mechanisms of nuclear membrane rupture and repair in both the main nucleus and micronuclei, and highlights recent work connecting the loss of nuclear integrity to genome instability and innate immune signaling. These connections link nuclear membrane rupture to complex chromosome alterations, tumorigenesis, and laminopathy etiologies.

8.
Nat Rev Mol Cell Biol ; 20(4): 259, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30816301

RESUMO

In the original Fig. 2a, telomeres are erroneously depicted having blunt ends following resection and CST-mediated fill-in. Instead, telomeres retain 3' overhangs, as depicted below.

9.
Curr Biol ; 28(21): 3422-3429.e5, 2018 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-30415700

RESUMO

The Mad1-Mad2 heterodimer is the catalytic hub of the spindle assembly checkpoint (SAC), which controls M phase progression through a multi-subunit anaphase inhibitor, the mitotic checkpoint complex (MCC) [1, 2]. During interphase, Mad1-Mad2 generates MCC at nuclear pores [3]. After nuclear envelope breakdown (NEBD), kinetochore-associated Mad1-Mad2 catalyzes MCC assembly until all chromosomes achieve bipolar attachment [1, 2]. Mad1-Mad2 and other factors are also incorporated into the fibrous corona, a phospho-dependent expansion of the outer kinetochore that precedes microtubule attachment [4-6]. The factor(s) involved in targeting Mad1-Mad2 to kinetochores in higher eukaryotes remain controversial [7-12], and the specific phosphorylation event(s) that trigger corona formation remain elusive [5, 13]. We used genome editing to eliminate Bub1, KNL1, and the Rod-Zw10-Zwilch (RZZ) complex in human cells. We show that RZZ's sole role in SAC activation is to tether Mad1-Mad2 to kinetochores. Separately, Mps1 kinase triggers fibrous corona formation by phosphorylating two N-terminal sites on Rod. In contrast, Bub1 and KNL1 activate kinetochore-bound Mad1-Mad2 to produce a "wait anaphase" signal but are not required for corona formation. We also show that clonal lines isolated after BUB1 disruption recover Bub1 expression and SAC function through nonsense-associated alternative splicing (NAS). Our study reveals a fundamental division of labor in the mammalian SAC and highlights a transcriptional response to nonsense mutations that can reduce or eliminate penetrance in genome editing experiments.


Assuntos
Cinetocoros/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Células HEK293 , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo
10.
Genome Med ; 10(1): 89, 2018 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-30482235

RESUMO

Telomere crisis is linked with many of the genomic alterations found in cancer genomes. A new understanding of how these alterations arise points towards an active role for innate immune sensors during crisis and to new opportunities for the treatment and diagnosis of cancer.


Assuntos
Neoplasias/genética , Telômero , Dano ao DNA , Genoma , Humanos
11.
Curr Biol ; 28(6): 872-883.e5, 2018 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-29502948

RESUMO

Monopolar spindle 1 (Mps1) is a conserved apical kinase in the spindle assembly checkpoint (SAC) that ensures accurate segregation of chromosomes during mitosis. Mps1 undergoes extensive auto- and transphosphorylation, but the regulatory and functional consequences of these modifications remain unclear. Recent findings highlight the importance of intermolecular interactions between the N-terminal extension (NTE) of Mps1 and the Hec1 subunit of the NDC80 complex, which control Mps1 localization at kinetochores and activation of the SAC. Whether the NTE regulates other mitotic functions of Mps1 remains unknown. Here, we report that phosphorylation within the NTE contributes to Mps1 activation through relief of catalytic autoinhibition that is mediated by the NTE itself. Moreover, we find that this regulatory NTE function is independent of its role in Mps1 kinetochore recruitment. We demonstrate that the NTE autoinhibitory mechanism impinges most strongly on Mps1-dependent SAC functions and propose that Mps1 activation likely occurs sequentially through dimerization of a "prone-to-autophosphorylate" Mps1 conformer followed by autophosphorylation of the NTE prior to maximal kinase activation segment trans-autophosphorylation. Our observations underline the importance of autoregulated Mps1 activity in generation and maintenance of a robust SAC in human cells.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas de Ciclo Celular/fisiologia , Proteínas do Citoesqueleto , Células HEK293 , Células HeLa , Humanos , Cinetocoros/fisiologia , Mitose , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Tirosina Quinases/fisiologia , Fuso Acromático/metabolismo
12.
Curr Opin Syst Biol ; 1: 54-61, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29177203

RESUMO

Cancer genome sequences contain footprints of somatic mutational processes, whose analysis in large tumor sequencing datasets has revealed novel mutational signatures, correlative features of variant topography, and complex events. Many of these analytic results have yet to reconciled with decades of mechanistic genome integrity research performed in controlled model systems. However, a new generation of genome-integrity experiments combining computational modeling, data analytics, and high-throughput sequencing are emerging to link mechanisms to patterns. Conversely, analytic studies evaluating quantitative footprints of specific genome integrity hypotheses will be critical in fitting naturally occurring mutational patterns to the predictions of a particular mechanistic model. Such quantitative and mechanistic studies will form the foundation of an emerging systems biology of genome integrity.

13.
Mol Cancer Res ; 15(11): 1579-1586, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28811362

RESUMO

The mammalian nuclear envelope (NE) forms a stable physical barrier between the nucleus and the cytoplasm, normally breaking down only during mitosis. However, spontaneous transient NE rupture in interphase can occur when NE integrity is compromised, such as when the nucleus experiences mechanical stress. For instance, deficiencies in the nuclear lamins and their associated proteins can cause NE rupture that is promoted by forces exerted by actin filaments. NE rupture can allow cytoplasmic nucleases to access chromatin, potentially compromising genome integrity. Importantly, spontaneous NE rupture was noted in several human cancer cell lines, but the cause of this defect is not known. Here, we investigated the mechanistic contributions of two major tumor suppressors, p53 (TP53) and Rb (RB1), to the repression of NE rupture. NE rupture was induced in normal human epithelial RPE-1 cells upon impairment of either Rb or p53 achieved by shRNA knockdown and CRISPR/Cas9 gene editing. NE rupture did not involve diminished expression of NE components or greater cell motility. However, cells that underwent NE rupture displayed a larger nuclear projection area. In conclusion, the data indicate that NE rupture in cancer cells is likely due to loss of either the Rb or the p53 pathway.Implications: These findings imply that tumor suppression by Rb and p53 includes the ability to prevent NE rupture, thereby protecting against genome alterations. Mol Cancer Res; 15(11); 1579-86. ©2017 AACR.


Assuntos
Membrana Nuclear/fisiologia , Proteínas de Ligação a Retinoblastoma/genética , Estresse Mecânico , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/genética , Sistemas CRISPR-Cas , Linhagem Celular , Movimento Celular , Células Epiteliais/citologia , Edição de Genes , Técnicas de Silenciamento de Genes , Humanos , Mitose , Neoplasias/genética
14.
Dev Cell ; 41(2): 143-156.e6, 2017 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-28441529

RESUMO

The spindle assembly checkpoint kinase Mps1 not only inhibits anaphase but also corrects erroneous attachments that could lead to missegregation and aneuploidy. However, Mps1's error correction-relevant substrates are unknown. Using a chemically tuned kinetochore-targeting assay, we show that Mps1 destabilizes microtubule attachments (K fibers) epistatically to Aurora B, the other major error-correcting kinase. Through quantitative proteomics, we identify multiple sites of Mps1-regulated phosphorylation at the outer kinetochore. Substrate modification was microtubule sensitive and opposed by PP2A-B56 phosphatases that stabilize chromosome-spindle attachment. Consistently, Mps1 inhibition rescued K-fiber stability after depleting PP2A-B56. We also identify the Ska complex as a key effector of Mps1 at the kinetochore-microtubule interface, as mutations that mimic constitutive phosphorylation destabilized K fibers in vivo and reduced the efficiency of the Ska complex's conversion from lattice diffusion to end-coupled microtubule binding in vitro. Our results reveal how Mps1 dynamically modifies kinetochores to correct improper attachments and ensure faithful chromosome segregation.


Assuntos
Segregação de Cromossomos/fisiologia , Cinetocoros/metabolismo , Metaloproteínas/metabolismo , Microtúbulos/metabolismo , Mitose/fisiologia , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/metabolismo , Anáfase/fisiologia , Aurora Quinase B/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/genética
15.
Nat Rev Mol Cell Biol ; 18(3): 175-186, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28096526

RESUMO

The shortening of human telomeres has two opposing effects during cancer development. On the one hand, telomere shortening can exert a tumour-suppressive effect through the proliferation arrest induced by activating the kinases ATM and ATR at unprotected chromosome ends. On the other hand, loss of telomere protection can lead to telomere crisis, which is a state of extensive genome instability that can promote cancer progression. Recent data, reviewed here, provide new evidence for the telomere tumour suppressor pathway and has revealed that telomere crisis can induce numerous cancer-relevant changes, including chromothripsis, kataegis and tetraploidization.


Assuntos
Instabilidade Genômica , Neoplasias/genética , Telômero/fisiologia , Cromotripsia , Humanos , Neoplasias/prevenção & controle , Telomerase/genética , Telomerase/metabolismo , Encurtamento do Telômero
16.
G3 (Bethesda) ; 6(8): 2343-54, 2016 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-27233670

RESUMO

In mammals the regulation of genomic instability plays a key role in tumor suppression and also controls genome plasticity, which is important for recombination during the processes of immunity and meiosis. Most studies to identify regulators of genomic instability have been performed in cells in culture or in systems that report on gross rearrangements of the genome, yet subtle differences in the level of genomic instability can contribute to whole organism phenotypes such as tumor predisposition. Here we performed a genome-wide association study in a population of 1379 outbred Crl:CFW(SW)-US_P08 mice to dissect the genetic landscape of micronucleus formation, a biomarker of chromosomal breaks, whole chromosome loss, and extranuclear DNA. Variation in micronucleus levels is a complex trait with a genome-wide heritability of 53.1%. We identify seven loci influencing micronucleus formation (false discovery rate <5%), and define candidate genes at each locus. Intriguingly at several loci we find evidence for sexual dimorphism in micronucleus formation, with a locus on chromosome 11 being specific to males.


Assuntos
Quebra Cromossômica , Estudo de Associação Genômica Ampla , Micronúcleos com Defeito Cromossômico , Locos de Características Quantitativas/genética , Animais , Mapeamento Cromossômico , Feminino , Instabilidade Genômica , Genótipo , Masculino , Camundongos , Fenótipo , Polimorfismo de Nucleotídeo Único , Caracteres Sexuais
17.
Cell ; 163(7): 1641-54, 2015 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-26687355

RESUMO

Telomere crisis occurs during tumorigenesis when depletion of the telomere reserve leads to frequent telomere fusions. The resulting dicentric chromosomes have been proposed to drive genome instability. Here, we examine the fate of dicentric human chromosomes in telomere crisis. We observed that dicentric chromosomes invariably persisted through mitosis and developed into 50-200 µm chromatin bridges connecting the daughter cells. Before their resolution at 3-20 hr after anaphase, the chromatin bridges induced nuclear envelope rupture in interphase, accumulated the cytoplasmic 3' nuclease TREX1, and developed RPA-coated single stranded (ss) DNA. CRISPR knockouts showed that TREX1 contributed to the generation of the ssDNA and the resolution of the chromatin bridges. Post-crisis clones showed chromothripsis and kataegis, presumably resulting from DNA repair and APOBEC editing of the fragmented chromatin bridge DNA. We propose that chromothripsis in human cancer may arise through TREX1-mediated fragmentation of dicentric chromosomes formed in telomere crisis.


Assuntos
Instabilidade Cromossômica , Cromossomos Humanos , Instabilidade Genômica , Neoplasias/genética , Telômero , Aberrações Cromossômicas , Citocinese , DNA de Cadeia Simples/metabolismo , Exodesoxirribonucleases/metabolismo , Humanos , Mitose , Membrana Nuclear/metabolismo , Fosfoproteínas/metabolismo
18.
Cell ; 156(5): 1017-31, 2014 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-24581499

RESUMO

The spindle assembly checkpoint (SAC) delays anaphase until all chromosomes are bioriented on the mitotic spindle. Under current models, unattached kinetochores transduce the SAC by catalyzing the intramitotic production of a diffusible inhibitor of APC/C(Cdc20) (the anaphase-promoting complex/cyclosome and its coactivator Cdc20, a large ubiquitin ligase). Here we show that nuclear pore complexes (NPCs) in interphase cells also function as scaffolds for anaphase-inhibitory signaling. This role is mediated by Mad1-Mad2 complexes tethered to the nuclear basket, which activate soluble Mad2 as a binding partner and inhibitor of Cdc20 in the cytoplasm. Displacing Mad1-Mad2 from nuclear pores accelerated anaphase onset, prevented effective correction of merotelic errors, and increased the threshold of kinetochore-dependent signaling needed to halt mitosis in response to spindle poisons. A heterologous Mad1-NPC tether restored Cdc20 inhibitor production and normal M phase control. We conclude that nuclear pores and kinetochores both emit "wait anaphase" signals that preserve genome integrity.


Assuntos
Anáfase , Proteínas de Ciclo Celular/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular , Proteínas Mad2/metabolismo , Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas de Ciclo Celular/genética , Dimerização , Células HCT116 , Células HeLa , Humanos , Interfase , Cinetocoros/metabolismo , Mitose , Proteínas Nucleares/genética
19.
Chromosoma ; 121(6): 565-72, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22566210

RESUMO

Polo-like kinase 1 (Plk1) is an essential mitotic regulator and undergoes periodic phosphorylation on threonine 210, a conserved residue in the kinase's activation loop. While phosphate-mimicking alterations of T210 stimulate Plk1's kinase activity in vitro, their effects on cell cycle regulation in vivo remain controversial. Using gene targeting, we replaced the native PLK1 locus in human cells with either PLK1 (T210A) or PLK1 (T210D) in both dominant and recessive settings. In contrast to previous reports, PLK1 (T210D) did not accelerate cells prematurely into mitosis, nor could it fulfill the kinase's essential role in chromosome congression. The latter was traced to an unexpected defect in Plk1-dependent phosphorylation of BubR1, a key mediator of stable kinetochore-microtubule attachment. Using chemical genetics to bypass this defect, we found that Plk1(T210D) is nonetheless able to induce equatorial RhoA zones and cleavage furrows during mitotic exit. Collectively, our data indicate that K-fibers are sensitive to even subtle perturbations in T210 phosphorylation and caution against relying on Plk1(T210D) as an in vivo surrogate for the natively activated kinase.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromossomos Humanos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Treonina/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular/efeitos dos fármacos , Posicionamento Cromossômico , Cromossomos Humanos/genética , Técnicas de Inativação de Genes , Humanos , Cinetocoros/metabolismo , Mitose , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Pirimidinas/farmacologia , Tionas/farmacologia , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
20.
J Cell Biol ; 190(1): 89-100, 2010 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-20624902

RESUMO

The spindle assembly checkpoint (SAC) in mammals uses cytosolic and kinetochore-based signaling pathways to inhibit anaphase. In this study, we use chemical genetics to show that the protein kinase Mps1 regulates both aspects of the SAC. Human MPS1-null cells were generated via gene targeting and reconstituted with either the wild-type kinase (Mps1(wt)) or a mutant version (Mps1(as)) sensitized to bulky purine analogues. Mps1 inhibition sharply accelerated anaphase onset, such that cells completed mitosis in 12 min, and prevented Cdc20's association with either Mad2 or BubR1 during interphase, i.e., before the appearance of functional kinetochores. Furthermore, intramitotic Mps1 inhibition evicted Bub1 and all other known SAC transducers from the outer kinetochore, but contrary to a recent study, did not perturb aurora B-dependent phosphorylation. We conclude that Mps1 has two complementary roles in SAC regulation: (1) initial cytoplasmic activation of Cdc20 inhibitors and (2) recruitment of factors that promote sustained anaphase inhibition and chromosome biorientation to unattached kinetochores.


Assuntos
Anáfase/fisiologia , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/fisiologia , Cromossomos Humanos/metabolismo , Interfase/fisiologia , Complexos Multiproteicos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Cdc20 , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Linhagem Celular Transformada , Cromossomos Humanos/genética , Humanos , Cinetocoros/metabolismo , Proteínas Mad2 , Complexos Multiproteicos/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo
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