RESUMO
Centrosome aberrations disrupt tissue architecture and may confer invasive properties to cancer cells. Here we show that structural centrosome aberrations, induced by overexpression of either Ninein-like protein (NLP) or CEP131/AZI1, sensitize polarized mammalian epithelia to basal cell extrusion. While unperturbed epithelia typically dispose of damaged cells through apical dissemination into luminal cavities, certain oncogenic mutations cause a switch in directionality towards basal cell extrusion, raising the potential for metastatic cell dissemination. Here we report that NLP-induced centrosome aberrations trigger the preferential extrusion of damaged cells towards the basal surface of epithelial monolayers. This switch in directionality from apical to basal dissemination coincides with a profound reorganization of the microtubule cytoskeleton, which in turn prevents the contractile ring repositioning that is required to support extrusion towards the apical surface. While the basal extrusion of cells harbouring NLP-induced centrosome aberrations requires exogenously induced cell damage, structural centrosome aberrations induced by excess CEP131 trigger the spontaneous dissemination of dying cells towards the basal surface from MDCK cysts. Thus, similar to oncogenic mutations, structural centrosome aberrations can favour basal extrusion of damaged cells from polarized epithelia. Assuming that additional mutations may promote cell survival, this process could sensitize epithelia to disseminate potentially metastatic cells.
Assuntos
Centrossomo/química , Células Epiteliais/citologia , Proteínas dos Microtúbulos/genética , Animais , Técnicas de Cultura de Células , Linhagem Celular , Polaridade Celular , Centrossomo/metabolismo , Cães , Células Epiteliais/metabolismo , Humanos , Células Madin Darby de Rim Canino , Proteínas dos Microtúbulos/metabolismo , MitoseRESUMO
Although aneuploidy is poorly tolerated during embryogenesis, aneuploidy and whole chromosomal instability (CIN) are common hallmarks of cancer, raising the question of how cancer cells can thrive in spite of chromosome aberrations. Here we present a comprehensive and quantitative proteomics analysis of isogenic DLD-1 colorectal adenocarcinoma cells lines, aimed at identifying cellular responses to changes in ploidy and/or CIN. Specifically, we compared diploid (2N) and tetraploid (4N) cells with posttetraploid aneuploid (PTA) clones and engineered trisomic clones. Our study provides a comparative data set on the proteomes and phosphoproteomes of the above cell lines, comprising several thousand proteins and phosphopeptides. In comparison to the parental 2N line, we observed changes in proteins associated with stress responses and with interferon signaling. Although we did not detect a conspicuous protein signature associated with CIN, we observed many changes in phosphopeptides that relate to fundamental cellular processes, including mitotic progression and spindle function. Most importantly, we found that most changes detectable in PTA cells were already present in the 4N progenitor line. This suggests that activation of mitotic pathways through hyper-phosphorylation likely constitutes an important response to chromosomal burden. In line with this conclusion, cells with extensive chromosome gains showed differential sensitivity toward a number of inhibitors targeting cell cycle kinases, suggesting that the efficacy of anti-mitotic drugs may depend on the karyotype of cancer cells.
Assuntos
Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Ploidias , Proteômica/métodos , Adenocarcinoma/genética , Aneuploidia , Linhagem Celular Tumoral/metabolismo , Instabilidade Cromossômica/genética , Instabilidade Cromossômica/fisiologia , Aberrações Cromossômicas , Neoplasias Colorretais/genética , Diploide , Humanos , Cariotipagem/métodos , Fosforilação , Proteoma/fisiologia , TetraploidiaRESUMO
Centrosomes are the main microtubule-organizing centers of animal cells. Although centrosome aberrations are common in tumors, their consequences remain subject to debate. Here, we studied the impact of structural centrosome aberrations, induced by deregulated expression of ninein-like protein (NLP), on epithelial spheres grown in Matrigel matrices. We demonstrate that NLP-induced structural centrosome aberrations trigger the escape ("budding") of living cells from epithelia. Remarkably, all cells disseminating into the matrix were undergoing mitosis. This invasive behavior reflects a novel mechanism that depends on the acquisition of two distinct properties. First, NLP-induced centrosome aberrations trigger a re-organization of the cytoskeleton, which stabilizes microtubules and weakens E-cadherin junctions during mitosis. Second, atomic force microscopy reveals that cells harboring these centrosome aberrations display increased stiffness. As a consequence, mitotic cells are pushed out of mosaic epithelia, particularly if they lack centrosome aberrations. We conclude that centrosome aberrations can trigger cell dissemination through a novel, non-cell-autonomous mechanism, raising the prospect that centrosome aberrations contribute to the dissemination of metastatic cells harboring normal centrosomes.
Assuntos
Centrossomo/metabolismo , Mitose , Neoplasias/metabolismo , Animais , Linhagem Celular Tumoral , Centrossomo/patologia , Cães , Epitélio/metabolismo , Epitélio/patologia , Humanos , Células Madin Darby de Rim Canino , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Metástase Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMO
Deregulation of centriole duplication has been implicated in cancer and primary microcephaly. Accordingly, it is important to understand how key centriole duplication factors are regulated. E3 ubiquitin ligases have been implicated in controlling the levels of several duplication factors, including PLK4, STIL and SAS-6, but the precise mechanisms ensuring centriole homeostasis remain to be fully understood. Here, we have combined proteomics approaches with the use of MLN4924, a generic inhibitor of SCF E3 ubiquitin ligases, to monitor changes in the cellular abundance of centriole duplication factors. We identified human STIL as a novel substrate of SCF-ßTrCP. The binding of ßTrCP depends on a DSG motif within STIL, and serine 395 within this motif is phosphorylated in vivo SCF-ßTrCP-mediated degradation of STIL occurs throughout interphase and mutations in the DSG motif causes massive centrosome amplification, attesting to the physiological importance of the pathway. We also uncover a connection between this new pathway and CDK2, whose role in centriole biogenesis remains poorly understood. We show that CDK2 activity protects STIL against SCF-ßTrCP-mediated degradation, indicating that CDK2 and SCF-ßTrCP cooperate via STIL to control centriole biogenesis.
Assuntos
Centríolos/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Ciclopentanos/farmacologia , Células HEK293 , Homeostase , Humanos , Interfase , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mutação , Fosforilação , Proteólise , Proteômica , Pirimidinas/farmacologia , Proteínas Ligases SKP Culina F-Box/antagonistas & inibidores , Serina/metabolismoRESUMO
Centrioles are conserved microtubule-based organelles that form the core of the centrosome and act as templates for the formation of cilia and flagella. Centrioles have important roles in most microtubule-related processes, including motility, cell division and cell signalling. To coordinate these diverse cellular processes, centriole number must be tightly controlled. In cycling cells, one new centriole is formed next to each pre-existing centriole in every cell cycle. Advances in imaging, proteomics, structural biology and genome editing have revealed new insights into centriole biogenesis, how centriole numbers are controlled and how alterations in these processes contribute to diseases such as cancer and neurodevelopmental disorders. Moreover, recent work has uncovered the existence of surveillance pathways that limit the proliferation of cells with numerical centriole aberrations. Owing to this progress, we now have a better understanding of the molecular mechanisms governing centriole biogenesis, opening up new possibilities for targeting these pathways in the context of human disease.
Assuntos
Centríolos/fisiologia , Animais , Ciclo Celular/fisiologia , Centrossomo/fisiologia , Cílios/fisiologia , Humanos , Microtúbulos/fisiologia , Mitose/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Aneuploidy is a hallmark of most human tumors, but the molecular physiology of aneuploid cells is not well characterized. In this study, we screened cell surface biomarkers of approximately 300 proteins by multiparameter flow cytometry using multiple aneuploid model systems such as cell lines, patient samples, and mouse models. Several new biomarkers were identified with altered expression in aneuploid cells, including overexpression of the cellular prion protein CD230/PrPC and the immunosuppressive cell surface enzyme ecto-5'-nucleotidase CD73. Functional analyses associated these alterations with increased cellular stress. An increased number of CD73+ cells was observed in confluent cultures in aneuploid cells relative to their diploid counterparts. An elevated expression in CD230/PrPC was observed in serum-deprived cells in association with increased generation of reactive oxygen species. Overall, our work identified biomarkers of aneuploid karyotypes, which suggest insights into the underlying molecular physiology of aneuploid cells. Cancer Res; 77(11); 2914-26. ©2017 AACR.
Assuntos
5'-Nucleotidase/metabolismo , Aneuploidia , Proteínas Priônicas/metabolismo , Estresse Fisiológico/fisiologia , 5'-Nucleotidase/biossíntese , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Camundongos , Transdução de SinaisRESUMO
Centrosomes, the main microtubule-organizing centers in animal cells, are replicated exactly once during the cell division cycle to form the poles of the mitotic spindle. Supernumerary centrosomes can lead to aberrant cell division and have been causally linked to chromosomal instability and cancer. Here, we report that an increase in the number of mature centrosomes, generated by disrupting cytokinesis or forcing centrosome overduplication, triggers the activation of the PIDDosome multiprotein complex, leading to Caspase-2-mediated MDM2 cleavage, p53 stabilization, and p21-dependent cell cycle arrest. This pathway also restrains the extent of developmentally scheduled polyploidization by regulating p53 levels in hepatocytes during liver organogenesis. Taken together, the PIDDosome acts as a first barrier, engaging p53 to halt the proliferation of cells carrying more than one mature centrosome to maintain genome integrity.
Assuntos
Centrossomo/fisiologia , Genes p53/genética , Complexos Multiproteicos/metabolismo , Ativação Transcricional/genética , Células A549 , Animais , Proteína Adaptadora de Sinalização CRADD/metabolismo , Caspase 2/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Células Cultivadas , Centrossomo/patologia , Citocinese/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Humanos , Fígado/citologia , Fígado/embriologia , Camundongos , Organogênese/genéticaRESUMO
Centrosomes determine the disposition of microtubule networks and thereby contribute to regulate cell shape, polarity, and motility, as well as chromosome segregation during cell division. Additionally, centrioles, the core components of centrosomes, are required for the formation of cilia and flagella. Mutations in genes coding for centrosomal and centriolar proteins are responsible for several human diseases, foremost ciliopathies and developmental disorders resulting in small brains (primary microcephaly) or small body size (dwarfism). Moreover, a long-standing postulate implicates numerical and/or structural centrosome aberrations in the etiology of cancer. In this review, we will discuss recent work on the role of centrosome aberrations in the promotion of genome instability and the disruption of tissue architecture, two hallmarks of human cancers. We will emphasize recent studies on the impact of centrosome aberrations on the polarity of epithelial cells cultured in three-dimensional spheroid models. Collectively, the results from these in vitro systems suggest that different types of centrosome aberrations can promote invasive behavior through different pathways. Particularly exciting is recent evidence indicating that centrosome aberrations may trigger the dissemination of potentially metastatic cells through a non-cell-autonomous mechanism.
RESUMO
Centrioles are essential for the formation of centrosomes and cilia. While numerical and/or structural centrosomes aberrations are implicated in cancer, mutations in centriolar and centrosomal proteins are genetically linked to ciliopathies, microcephaly, and dwarfism. The evolutionarily conserved mechanisms underlying centrosome biogenesis are centered on a set of key proteins, including Plk4, Sas-6, and STIL, whose exact levels are critical to ensure accurate reproduction of centrioles during cell cycle progression. However, neither the intracellular levels of centrosomal proteins nor their stoichiometry within centrosomes is presently known. Here, we have used two complementary approaches, targeted proteomics and EGFP-tagging of centrosomal proteins at endogenous loci, to measure protein abundance in cultured human cells and purified centrosomes. Our results provide a first assessment of the absolute and relative amounts of major components of the human centrosome. Specifically, they predict that human centriolar cartwheels comprise up to 16 stacked hubs and 1 molecule of STIL for every dimer of Sas-6. This type of quantitative information will help guide future studies of the molecular basis of centrosome assembly and function.
Assuntos
Proteínas de Ciclo Celular/análise , Centrossomo/química , Imagem Óptica , Proteômica , Linhagem Celular , Células Epiteliais/química , HumanosRESUMO
The multiplexing capabilities of isobaric mass tag-based protein quantification, such as Tandem Mass Tags or Isobaric Tag for Relative and Absolute Quantitation have dramatically increased the scope of mass spectrometry-based proteomics studies. Not only does the technology allow for the simultaneous quantification of multiple samples in a single MS injection, but its seamless compatibility with extensive sample prefractionation methods allows for comprehensive studies of complex proteomes. However, reporter ion-based quantification has often been criticized for limited quantification accuracy due to interference from coeluting peptides and peptide fragments. In this study, we investigate the extent of this problem and propose an effective and easy-to-implement remedy that relies on spiking a 6-protein calibration mixture to the samples. We evaluated our ratio adjustment approach using two large scale TMT 10-plex data sets derived from a human cancer and noncancer cell line as well as E. coli cells grown at two different conditions. Furthermore, we analyzed a complex 2-proteome artificial sample mixture and investigated the precision of TMT and precursor ion intensity-based label free quantification. Studying the protein set identified by both methods, we found that differentially abundant proteins were assigned dramatically higher statistical significance when quantified using TMT. Data are available via ProteomeXchange with identifier PXD003346.
Assuntos
Proteoma/análise , Proteômica/métodos , Linhagem Celular , Linhagem Celular Tumoral , Interpretação Estatística de Dados , Escherichia coli , Humanos , Proteoma/normas , Proteômica/normas , Espectrometria de Massas em Tandem/métodosRESUMO
PICH is a SNF2 family DNA translocase that binds to ultra-fine DNA bridges (UFBs) in mitosis. Numerous roles for PICH have been proposed from protein depletion experiments, but a consensus has failed to emerge. Here, we report that deletion of PICH in avian cells causes chromosome structural abnormalities, and hypersensitivity to an inhibitor of Topoisomerase II (Topo II), ICRF-193. ICRF-193-treated PICH(-/-) cells undergo sister chromatid non-disjunction in anaphase, and frequently abort cytokinesis. PICH co-localizes with Topo IIα on UFBs and at the ribosomal DNA locus, and the timely resolution of both structures depends on the ATPase activity of PICH. Purified PICH protein strongly stimulates the catalytic activity of Topo II in vitro. Consistent with this, a human PICH(-/-) cell line exhibits chromosome instability and chromosome condensation and decatenation defects similar to those of ICRF-193-treated cells. We propose that PICH and Topo II cooperate to prevent chromosome missegregation events in mitosis.
Assuntos
Antígenos de Neoplasias/metabolismo , Proteínas Aviárias/genética , Proteínas de Ciclo Celular/genética , Cromátides/metabolismo , Segregação de Cromossomos/genética , DNA Helicases/genética , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mitose/genética , Animais , Proteínas Aviárias/metabolismo , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Galinhas , Instabilidade Cromossômica/genética , DNA Helicases/metabolismo , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Técnicas de Inativação de Genes , Humanos , Linfócitos/metabolismoRESUMO
During mitosis, Bub1 kinase phosphorylates histone H2A-T120 to promote centromere sister chromatid cohesion through recruitment of shugoshin (Sgo) proteins. The regulation and dynamics of H2A-T120 phosphorylation are poorly understood. Using quantitative phosphoproteomics we show that Bub1 is autophosphorylated at numerous sites. We confirm mitosis-specific autophosphorylation of a several residues and show that Bub1 activation is primed in interphase but fully achieved only in mitosis. Mutation of a single autophosphorylation site T589 alters kinetochore turnover of Bub1 and results in uniform H2A-T120 phosphorylation and Sgo recruitment along chromosome arms. Consequently, improper sister chromatid resolution and chromosome segregation errors are observed. Kinetochore tethering of Bub1-T589A refocuses H2A-T120 phosphorylation and Sgo1 to centromeres. Recruitment of the Bub1-Bub3-BubR1 axis to kinetochores has recently been extensively studied. Our data provide novel insight into the regulation and kinetochore residency of Bub1 and indicate that its localization is dynamic and tightly controlled through feedback autophosphorylation.
Assuntos
Retroalimentação Fisiológica , Histonas/metabolismo , Cinetocoros/metabolismo , Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Centrômero/metabolismo , Cromatografia Líquida , Imunofluorescência , Células HeLa , Humanos , Interfase , Espectrometria de Massas , Microscopia Confocal , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose , Espectrometria de Massas em TandemRESUMO
Equal mitotic chromosome segregation is critical for genome integrity and is monitored by the spindle assembly checkpoint (SAC). We have previously shown that the consensus phosphorylation motif of the essential SAC kinase Monopolar spindle 1 (Mps1) is very similar to that of Polo-like kinase 1 (Plk1). This prompted us to ask whether human Plk1 cooperates with Mps1 in SAC signaling. Here, we demonstrate that Plk1 promotes checkpoint signaling at kinetochores through the phosphorylation of at least two Mps1 substrates, including KNL-1 and Mps1 itself. As a result, Plk1 activity enhances Mps1 catalytic activity as well as the recruitment of the SAC components Mad1:C-Mad2 and Bub3:BubR1 to kinetochores. We conclude that Plk1 strengthens the robustness of SAC establishment at the onset of mitosis and supports SAC maintenance during prolonged mitotic arrest.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular , Humanos , Proteínas Mad2/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Fuso Acromático/metabolismo , Quinase 1 Polo-LikeRESUMO
Centrioles function as core components of centrosomes and as basal bodies for the formation of cilia and flagella. Thus, effective control of centriole numbers is essential for embryogenesis, tissue homeostasis and genome stability. In mammalian cells, the centriole duplication cycle is governed by Polo-like kinase 4 (Plk4). Here, we identify the E3 ubiquitin ligase Mind bomb (Mib1) as a new interaction partner of Plk4. We show that Mib1 localizes to centriolar satellites but redistributes to centrioles in response to conditions that induce centriole amplification. The E3 ligase activity of Mib1 triggers ubiquitylation of Plk4 on multiple sites, causing the formation of Lys11-, Lys29- and Lys48-ubiquitin linkages. These modifications control the abundance of Plk4 and its ability to interact with centrosomal proteins, thus counteracting centriole amplification induced by excess Plk4. Collectively, these results identify the interaction between Mib1 and Plk4 as a new and important element in the control of centriole homeostasis.
Assuntos
Centríolos/metabolismo , Biogênese de Organelas , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Ligação Proteica , UbiquitinaçãoRESUMO
Centrioles function in the assembly of centrosomes and cilia. Structural and numerical centrosome aberrations have long been implicated in cancer, and more recent genetic evidence directly links centrosomal proteins to the etiology of ciliopathies, dwarfism and microcephaly. To better understand these disease connections, it will be important to elucidate the biogenesis of centrioles as well as the controls that govern centriole duplication during the cell cycle. Moreover, it remains to be fully understood how these organelles organize a variety of dynamic microtubule-based structures in response to different physiological conditions. In proliferating cells, centrosomes are crucial for the assembly of microtubule arrays, including mitotic spindles, whereas in quiescent cells centrioles function as basal bodies in the formation of ciliary axonemes. In this short review, we briefly introduce the key gene products required for centriole duplication. Then we discuss recent findings on the centriole duplication factor STIL that point to centrosome amplification as a potential root cause for primary microcephaly in humans. We also present recent data on the role of a disease-related centriole-associated protein complex, Cep164-TTBK2, in ciliogenesis.
Assuntos
Centríolos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microcefalia/genética , Animais , Proteínas de Ciclo Celular , Centríolos/genética , Centríolos/patologia , Proteínas do Citoesqueleto , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
The Plk1-interacting checkpoint helicase (PICH) protein localizes to ultrafine anaphase bridges (UFBs) in mitosis alongside a complex of DNA repair proteins, including the Bloom's syndrome protein (BLM). However, very little is known about the function of PICH or how it is recruited to UFBs. Using a combination of microfluidics, fluorescence microscopy, and optical tweezers, we have defined the properties of PICH in an in vitro model of an anaphase bridge. We show that PICH binds with a remarkably high affinity to duplex DNA, resulting in ATP-dependent protein translocation and extension of the DNA. Most strikingly, the affinity of PICH for binding DNA increases with tension-induced DNA stretching, which mimics the effect of the mitotic spindle on a UFB. PICH binding also appears to diminish force-induced DNA melting. We propose a model in which PICH recognizes and stabilizes DNA under tension during anaphase, thereby facilitating the resolution of entangled sister chromatids.
Assuntos
Anáfase/genética , DNA Helicases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cromátides/metabolismo , DNA Helicases/química , DNA Helicases/genética , Humanos , Microscopia de Fluorescência/métodos , Ácidos Nucleicos Heteroduplexes/metabolismo , Nucleossomos/metabolismo , Transporte Proteico , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Intraflagellar transport (IFT) of ciliary precursors such as tubulin from the cytoplasm to the ciliary tip is involved in the construction of the cilium, a hairlike organelle found on most eukaryotic cells. However, the molecular mechanisms of IFT are poorly understood. Here, we found that the two core IFT proteins IFT74 and IFT81 form a tubulin-binding module and mapped the interaction to a calponin homology domain of IFT81 and a highly basic domain in IFT74. Knockdown of IFT81 and rescue experiments with point mutants showed that tubulin binding by IFT81 was required for ciliogenesis in human cells.
Assuntos
Cílios/fisiologia , Proteínas do Citoesqueleto/metabolismo , Proteínas Musculares/metabolismo , Tubulina (Proteína)/metabolismo , Linhagem Celular Tumoral , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Cílios/genética , Cristalografia por Raios X , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Técnicas de Silenciamento de Genes , Humanos , Proteínas Musculares/química , Proteínas Musculares/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Mutação Puntual , Estrutura Terciária de Proteína , Transporte Proteico , RNA Interferente Pequeno/genéticaRESUMO
Polo-like kinase 1 (Plk1) controls multiple aspects of mitosis and is activated through its phosphorylation at Thr210. Here we identify Ser99 on Plk1 as a novel mitosis-specific phosphorylation site, which operates independently of Plk1-Thr210 phosphorylation. Plk1-Ser99 phosphorylation creates a docking site for 14-3-3γ, and this interaction stimulates the catalytic activity of Plk1. Knockdown of 14-3-3γ or replacement of wild-type (WT) Plk1 by a Ser99-phospho-blocking mutant leads to a prometaphase/metaphase-like arrest due to the activation of the spindle assembly checkpoint. Inhibition of phosphatidylinositol 3-kinase (PI3K) and Akt significantly reduces the level of Plk1-Ser99 phosphorylation and delays metaphase to anaphase transition. Plk1-Ser99 phosphorylation requires not only Akt activity but also protein(s) associated with Plk1 in a mitosis-specific manner. Therefore, mitotic Plk1 activity is regulated not only by Plk1-Thr210 phosphorylation, but also by Plk1 binding to 14-3-3γ following Plk1-Ser99 phosphorylation downstream of the PI3K-Akt signalling pathway. This novel Plk1 activation pathway controls proper progression from metaphase to anaphase.
Assuntos
Proteínas 14-3-3/metabolismo , Anáfase , Proteínas de Ciclo Celular/metabolismo , Metáfase , Fosfatidilinositol 3-Quinases/metabolismo , Fosfosserina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Biocatálise , Caenorhabditis elegans , Drosophila melanogaster , Ativação Enzimática , Células HeLa , Humanos , Pontos de Checagem da Fase M do Ciclo Celular , Modelos Biológicos , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Quinase 1 Polo-LikeAssuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Animais , Humanos , Invertebrados/genética , Invertebrados/metabolismo , Meiose , Mitose , Plantas/genética , Plantas/metabolismo , Vertebrados/genética , Vertebrados/metabolismo , Leveduras/genética , Leveduras/metabolismo , Quinase 1 Polo-LikeRESUMO
Centrioles organize the centrosome, and accurate control of their number is critical for the maintenance of genomic integrity. Centriole duplication occurs once per cell cycle and is controlled by Polo-like kinase 4 (Plk4). We showed previously that Plk4 phosphorylates itself to promote its degradation by the proteasome. Here we demonstrate that this autoregulated instability controls the abundance of endogenous Plk4. Preventing Plk4 autoregulation causes centrosome amplification, stabilization of p53, and loss of cell proliferation; moreover, suppression of p53 allows growth of cells carrying amplified centrosomes. Plk4 autoregulation thus guards against genome instability by limiting centrosome duplication to once per cell cycle.