RESUMEN
Eukaryotic genomes replicate via spatially and temporally regulated origin firing. Cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) promote origin firing, whereas the S phase checkpoint limits firing to prevent nucleotide and RPA exhaustion. We used chemical genetics to interrogate human DDK with maximum precision, dissect its relationship with the S phase checkpoint, and identify DDK substrates. We show that DDK inhibition (DDKi) leads to graded suppression of origin firing and fork arrest. S phase checkpoint inhibition rescued origin firing in DDKi cells and DDK-depleted Xenopus egg extracts. DDKi also impairs RPA loading, nascent-strand protection, and fork restart. Via quantitative phosphoproteomics, we identify the BRCA1-associated (BRCA1-A) complex subunit MERIT40 and the cohesin accessory subunit PDS5B as DDK effectors in fork protection and restart. Phosphorylation neutralizes autoinhibition mediated by intrinsically disordered regions in both substrates. Our results reveal mechanisms through which DDK controls the duplication of large vertebrate genomes.
Asunto(s)
Replicación del ADN , Origen de Réplica , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Puntos de Control de la Fase S del Ciclo Celular , Especificidad por Sustrato , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Xenopus laevisRESUMEN
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.
Asunto(s)
Cinetocoros/metabolismo , Puntos de Control de la Fase M del Ciclo Celular/fisiología , Células HEK293 , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Puntos de Control de la Fase M del Ciclo Celular/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas de Ciclo Celular/fisiología , Proteínas del Citoesqueleto , Células HEK293 , Células HeLa , Humanos , Cinetocoros/fisiología , Mitosis , Proteínas Nucleares/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Tirosina Quinasas/fisiología , Huso Acromático/metabolismoRESUMEN
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.
Asunto(s)
Segregación Cromosómica/fisiología , Cinetocoros/metabolismo , Metaloproteínas/metabolismo , Microtúbulos/metabolismo , Mitosis/fisiología , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Ribosómicas/metabolismo , Anafase/fisiología , Aurora Quinasa B/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Humanos , Puntos de Control de la Fase M del Ciclo Celular/genéticaRESUMEN
PLK4 is the major kinase driving centriole duplication. Duplication occurs only once per cell cycle, forming one new (or daughter) centriole that is tightly engaged to the preexisting (or mother) centriole. Centriole engagement is known to block the reduplication of mother centrioles, but the molecular identity responsible for the block remains unclear. Here, we show that the centriolar cartwheel, the geometric scaffold for centriole assembly, forms the identity of daughter centrioles essential for the block, ceasing further duplication of the mother centriole to which it is engaged. To ensure a steady block, we found that the cartwheel requires constant maintenance by PLK4 through phosphorylation of the same substrate that drives centriole assembly, revealing a parsimonious control in which "assembly" and "block for new assembly" are linked through the same catalytic reaction to achieve homeostasis. Our results support a recently deduced model that the cartwheel-bound PLK4 directly suppresses centriole reduplication.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Centriolos/fisiología , Homeostasis/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Ciclo Celular/fisiología , Humanos , Fosforilación/fisiologíaRESUMEN
During mitosis, multiple protein kinases transform the cytoskeleton and chromosomes into new and highly dynamic structures that mediate the faithful transmission of genetic information and cell division. However, the large number and strong conservation of mammalian kinases in general pose significant obstacles to interrogating them with small molecules, due to the difficulty in identifying and validating those which are truly selective. To overcome this problem, a steric complementation strategy has been developed, in which a bulky "gatekeeper" residue within the active site of the kinase of interest is replaced with a smaller amino acid, such as glycine or alanine. The enlarged catalytic pocket can then be targeted in an allele-specific manner with bulky purine analogs. This strategy provides a general framework for dissecting kinase function with high selectivity, rapid kinetics, and reversibility. In this chapter we discuss the principles and techniques needed to implement this chemical genetic approach in mammalian cells.
Asunto(s)
Mitosis , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Ingeniería de Proteínas , Alelos , Animales , Línea Celular , Clonación Molecular , Edición Génica , Puntos de Control de la Fase M del Ciclo Celular/genética , Mitosis/genética , Mutagénesis , Penetrancia , Fosfotransferasas/química , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The cohesin complex links DNA molecules and plays key roles in the organization, expression, repair, and segregation of eukaryotic genomes. In vertebrates the Esco1 and Esco2 acetyltransferases both modify cohesin's Smc3 subunit to establish sister chromatid cohesion during S phase, but differ in their N-terminal domains and expression during development and across the cell cycle. Here we show that Esco1 and Esco2 also differ dramatically in their interaction with chromatin, as Esco1 is recruited by cohesin to over 11,000 sites, whereas Esco2 is infrequently enriched at REST/NRSF target genes. Esco1's colocalization with cohesin occurs throughout the cell cycle and depends on two short motifs (the A-box and B-box) present in and unique to all Esco1 orthologs. Deleting either motif led to the derepression of Esco1-proximal genes and functional uncoupling of cohesion from Smc3 acetylation. In contrast, other mutations that preserved Esco1's recruitment separated its roles in cohesion establishment and gene silencing. We conclude that Esco1 uses cohesin as both a substrate and a scaffold for coordinating multiple chromatin-based transactions in somatic cells.
Asunto(s)
Acetiltransferasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Regulación de la Expresión Génica , Genoma Humano , Acetiltransferasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión/genética , Western Blotting , Proteínas de Ciclo Celular/genética , Cromátides/genética , Cromátides/metabolismo , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/genética , Células HCT116 , Células HeLa , Humanos , Datos de Secuencia Molecular , Unión Proteica , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Transcripción Genética , CohesinasRESUMEN
Excess dormant origins bound by the minichromosome maintenance (MCM) replicative helicase complex play a critical role in preventing replication stress, chromosome instability, and tumorigenesis. In response to DNA damage, replicating cells must coordinate DNA repair and dormant origin firing to ensure complete and timely replication of the genome; how cells regulate this process remains elusive. Herein, we identify a member of the Fanconi anemia (FA) DNA repair pathway, FANCI, as a key effector of dormant origin firing in response to replication stress. Cells lacking FANCI have reduced number of origins, increased inter-origin distances, and slowed proliferation rates. Intriguingly, ATR-mediated FANCI phosphorylation inhibits dormant origin firing while promoting replication fork restart/DNA repair. Using super-resolution microscopy, we show that FANCI co-localizes with MCM-bound chromatin in response to replication stress. These data reveal a unique role for FANCI as a modulator of dormant origin firing and link timely genome replication to DNA repair.
Asunto(s)
Cromatina/metabolismo , Daño del ADN , Replicación del ADN , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Proliferación Celular , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Células HeLa , Humanos , Hidroxiurea/farmacología , Proteínas de Mantenimiento de Minicromosoma/genética , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de SeñalRESUMEN
An intercentrosomal linker keeps a cell's two centrosomes joined together until it is dissolved at the onset of mitosis. A second connection keeps daughter centrioles engaged to their mothers until they lose their orthogonal arrangement at the end of mitosis. Centriole disengagement is required to license centrioles for duplication. We show that the intercentrosomal linker protein Cep68 is degraded in prometaphase through the SCF(ßTrCP) (Skp1-Cul1-F-box protein) ubiquitin ligase complex. Cep68 degradation is initiated by PLK1 phosphorylation of Cep68 on Ser 332, allowing recognition by ßTrCP. We also found that Cep68 forms a complex with Cep215 (also known as Cdk5Rap2) and PCNT (also known as pericentrin), two PCM (pericentriolar material) proteins involved in centriole engagement. Cep68 and PCNT bind to different pools of Cep215. We propose that Cep68 degradation allows Cep215 removal from the peripheral PCM preventing centriole separation following disengagement, whereas PCNT cleavage mediates Cep215 removal from the core of the PCM to inhibit centriole disengagement and duplication.
Asunto(s)
Antígenos/metabolismo , Centriolos/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteolisis , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Metafase/genética , Fosforilación , Prometafase/genética , Unión Proteica , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , Proteínas Ligasas SKP Cullina F-box/genética , Quinasa Tipo Polo 1RESUMEN
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.
Asunto(s)
Anafase , Proteínas de Ciclo Celular/metabolismo , Puntos de Control de la Fase M del Ciclo Celular , Proteínas Mad2/metabolismo , Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Transporte Activo de Núcleo Celular , Proteínas de Ciclo Celular/genética , Dimerización , Células HCT116 , Células HeLa , Humanos , Interfase , Cinetocoros/metabolismo , Mitosis , Proteínas Nucleares/genéticaRESUMEN
During oncogenesis, cells acquire multiple genetic alterations that confer essential tumor-specific traits, including immortalization, escape from antimitogenic signaling, neovascularization, invasiveness, and metastatic potential. In most instances, these alterations are thought to arise incrementally over years, if not decades. However, recent progress in sequencing cancer genomes has begun to challenge this paradigm, because a radically different phenomenon, termed chromothripsis, has been suggested to cause complex intra- and interchromosomal rearrangements on short timescales. In this Review, we review established pathways crucial for genome integrity and discuss how their dysfunction could precipitate widespread chromosome breakage and rearrangement in the course of malignancy.
Asunto(s)
Aberraciones Cromosómicas , Neoplasias/etiología , Neoplasias/genética , Animales , Apoptosis , Inestabilidad Cromosómica , Rotura Cromosómica , Reparación del ADN , Replicación del ADN , Reordenamiento Génico , Humanos , Mitosis , Modelos Genéticos , Mutación , Acortamiento del TelómeroRESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromosomas Humanos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Treonina/metabolismo , Proteínas de Ciclo Celular/genética , Línea Celular/efectos de los fármacos , Posicionamiento de Cromosoma , Cromosomas Humanos/genética , Técnicas de Inactivación de Genes , Humanos , Cinetocoros/metabolismo , Mitosis , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Pirimidinas/farmacología , Tionas/farmacología , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismo , Quinasa Tipo Polo 1RESUMEN
Polo-like kinase 1 (Plk1) is a core regulator of cell division and an emerging target for cancer therapy. Pharmacologic inhibitors of Plk1 exist but affect other kinases, complicating their in vivo validation. To address this, we examined effects of two structurally unrelated Plk1 inhibitors (BI-2536 and TAL) against isogenic human cell lines that solely express wildtype (wt) or analogue-sensitive (as) Plk1 alleles. Unexpectedly, Plk1(as) cells displayed profound biochemical and functional resistance to both inhibitors. Cells that co-express Plk1(wt) and Plk1(as) exhibit loss-of-function phenotypes only when both kinase alleles are inhibited. Resistance to BI-2536 is linked to an intragenic suppressor mutation (C67V) that restores an otherwise invariant valine to the kinase active site. Structural modeling demonstrates that this mutation not only enables Plk1(as) to function in vivo but also occludes BI-2536 from the ATP-binding pocket. Our results reveal the molecular basis of Plk inhibitor selectivity and a potential mechanism for tumor cell resistance.
Asunto(s)
Compuestos de Anilina/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Resistencia a Medicamentos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Pteridinas/farmacología , Tiazolidinas/farmacología , Compuestos de Anilina/química , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Humanos , Mutación , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Pteridinas/química , Tiazolidinas/química , Quinasa Tipo Polo 1RESUMEN
Delineation of phosphorylation-based signaling networks requires reliable data about the underlying cellular kinase-substrate interactions. We report a chemical genetics and quantitative phosphoproteomics approach that encompasses cellular kinase activation in combination with comparative replicate mass spectrometry analyses of cells expressing either inhibitor-sensitive or resistant kinase variant. We applied this workflow to Plk1 (Polo-like kinase 1) in mitotic cells and induced cellular Plk1 activity by wash-out of the bulky kinase inhibitor 3-MB-PP1, which targets a mutant kinase version with an enlarged catalytic pocket while not interfering with wild-type Plk1. We quantified more than 20,000 distinct phosphorylation sites by SILAC, approximately half of which were measured in at least two independent experiments in cells expressing mutant and wild-type Plk1. Based on replicate phosphorylation site quantifications in both mutant and wild-type Plk1 cells, our chemical genetic proteomics concept enabled stringent comparative statistics by significance analysis of microarrays, which unveiled more than 350 cellular downstream targets of Plk1 validated by full concordance of both statistical and experimental data. Our data point to hitherto poorly characterized aspects in Plk1-controlled mitotic progression and provide a largely extended resource for functional studies. We anticipate the described strategies to be of general utility for systematic and confident identification of cellular protein kinase substrates.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteómica/métodos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de Ciclo Celular/genética , Línea Celular , Humanos , Mutación , Fosforilación , Análisis por Matrices de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Transducción de Señal , Quinasa Tipo Polo 1RESUMEN
The ring-shaped cohesin complex links sister chromatids and plays crucial roles in homologous recombination and mitotic chromosome segregation. In cycling cells, cohesin's ability to generate cohesive linkages is restricted to S phase and depends on loading and establishment factors that are intimately connected to DNA replication. Here we review how cohesin is regulated by the replication machinery, as well as recent evidence that cohesin itself influences how chromosomes are replicated.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Proteínas Cromosómicas no Histona/fisiología , Replicación del ADN , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas , CohesinasRESUMEN
Targeted therapies for cancer promise to revolutionize treatment by specifically inactivating pathways needed for the growth of tumor cells. The most prominent example of such therapy is imatinib (Gleevec), which targets the BCR-ABL kinase and provides an effective low-toxicity treatment for chronic myelogenous leukemia. This success has spawned myriad efforts to develop similarly targeted drugs for other cancers. Unfortunately, the high expectations of these efforts have not yet been realized, likely due to the genetic diversity among and within tumors, as well as the complex and largely unpredictable interactions of drug-like compounds with innumerable targets that affect cellular and organismal metabolism. While improvements in sequencing technologies are beginning to address the first problem, solving the second problem requires methods for linking specific features of the cancer genome to their optimally targeted therapies. One approach, referred to as chemical genetics, accomplishes this by genetic control of chemical susceptibility. Chemical genetics is a crucial tool for the rational development of cancer drugs.
Asunto(s)
Antineoplásicos/farmacología , Descubrimiento de Drogas , Animales , Resistencia a Antineoplásicos , Genes ras , Humanos , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Transducción de SeñalRESUMEN
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.
Asunto(s)
Anafase/fisiología , Proteínas de Ciclo Celular/metabolismo , División Celular/fisiología , Cromosomas Humanos/metabolismo , Interfase/fisiología , Complejos Multiproteicos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/fisiología , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas Cdc20 , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular Transformada , Cromosomas Humanos/genética , Humanos , Cinetocoros/metabolismo , Proteínas Mad2 , Complejos Multiproteicos/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
Cohesin not only links sister chromatids but also inhibits the transcriptional machinery's interaction with and movement along chromatin. In contrast, replication forks must traverse such cohesin-associated obstructions to duplicate the entire genome in S phase. How this occurs is unknown. Through single-molecule analysis, we demonstrate that the replication factor C (RFC)-CTF18 clamp loader (RFC(CTF18)) controls the velocity, spacing and restart activity of replication forks in human cells and is required for robust acetylation of cohesin's SMC3 subunit and sister chromatid cohesion. Unexpectedly, we discovered that cohesin acetylation itself is a central determinant of fork processivity, as slow-moving replication forks were found in cells lacking the Eco1-related acetyltransferases ESCO1 or ESCO2 (refs 8-10) (including those derived from Roberts' syndrome patients, in whom ESCO2 is biallelically mutated) and in cells expressing a form of SMC3 that cannot be acetylated. This defect was a consequence of cohesin's hyperstable interaction with two regulatory cofactors, WAPL and PDS5A (refs 12, 13); removal of either cofactor allowed forks to progress rapidly without ESCO1, ESCO2, or RFC(CTF18). Our results show a novel mechanism for clamp-loader-dependent fork progression, mediated by the post-translational modification and structural remodelling of the cohesin ring. Loss of this regulatory mechanism leads to the spontaneous accrual of DNA damage and may contribute to the abnormalities of the Roberts' syndrome cohesinopathy.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Replicación del ADN/fisiología , ATPasas Asociadas con Actividades Celulares Diversas , Acetilación , Acetiltransferasas/deficiencia , Acetiltransferasas/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/química , Línea Celular , Senescencia Celular , Cromátides/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/deficiencia , Proteínas Cromosómicas no Histona/genética , Daño del ADN , Replicación del ADN/efectos de los fármacos , Humanos , Mutágenos/toxicidad , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Subunidades de Proteína/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteína de Replicación C/metabolismo , CohesinasRESUMEN
The integrity of genomic DNA is continuously challenged by the presence of DNA base lesions or DNA strand breaks. Here we report the identification of a new DNA damage response protein, SMARCAL1 (SWI/SNF-related, matrix associated, actin-dependent regulator of chromatin, subfamily a-like 1), which is a member of the SNF2 family and is mutated in Schimke immunoosseous dysplasia (SIOD). We demonstrate that SMARCAL1 directly interacts with Replication protein A (RPA) and is recruited to sites of DNA damage in an RPA-dependent manner. SMARCAL1-depleted cells display sensitivity to DNA-damaging agents that induce replication fork collapse, and exhibit slower fork recovery and delayed entry into mitosis following S-phase arrest. Furthermore, SIOD patient fibroblasts reconstituted with SMARCAL1 exhibit faster cell cycle progression after S-phase arrest. Thus, the symptoms of SIOD may be caused, at least in part, by defects in the cellular response to DNA replication stress.
Asunto(s)
ADN Helicasas/metabolismo , Osteocondrodisplasias/fisiopatología , Proteína de Replicación A/metabolismo , Secuencia de Aminoácidos , Ciclo Celular , Línea Celular , Daño del ADN , ADN Helicasas/química , Replicación del ADN , Humanos , Datos de Secuencia Molecular , Osteocondrodisplasias/genética , Alineación de SecuenciaRESUMEN
It has been proposed that separase-dependent centriole disengagement at anaphase licenses centrosomes for duplication in the next cell cycle. Here we test whether such a mechanism exists in intact human cells. Loss of separase blocked centriole disengagement during mitotic exit and delayed assembly of new centrioles during the following S phase; however, most engagements were eventually dissolved. We identified Polo-like kinase 1 (Plk1) as a parallel activator of centriole disengagement. Timed inhibition of Plk1 mapped its critical period of action to late G2 or early M phase, i.e., prior to securin destruction and separase activation at anaphase onset. Crucially, when cells exited mitosis after downregulation of both separase and Plk1, centriole disengagement failed completely, and subsequent centriole duplication in interphase was also blocked. Our results indicate that Plk1 and separase act at different times during M phase to license centrosome duplication, reminiscent of their roles in removing cohesin from chromosomes.