RESUMO
The Spindle Assembly Checkpoint (SAC) ensures genomic stability by preventing sister chromatid separation until all chromosomes are attached to the spindle. It catalyzes the production of the Mitotic Checkpoint Complex (MCC), which inhibits Cdc20 to inactivate the Anaphase Promoting Complex/Cyclosome (APC/C). Here we show that two Cdc20-binding motifs in BubR1 of the recently identified ABBA motif class are crucial for the MCC to recognize active APC/C-Cdc20. Mutating these motifs eliminates MCC binding to the APC/C, thereby abolishing the SAC and preventing cells from arresting in response to microtubule poisons. These ABBA motifs flank a KEN box to form a cassette that is highly conserved through evolution, both in the arrangement and spacing of the ABBA-KEN-ABBA motifs, and association with the amino-terminal KEN box required to form the MCC. We propose that the ABBA-KEN-ABBA cassette holds the MCC onto the APC/C by binding the two Cdc20 molecules in the MCC-APC/C complex.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/genética , Proteínas Cdc20/genética , Pontos de Checagem da Fase M do Ciclo Celular , Proteínas Serina-Treonina Quinases/genética , Motivos de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase/química , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Evolução Biológica , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas Cdc20/química , Proteínas Cdc20/metabolismo , Ciona intestinalis/genética , Ciona intestinalis/metabolismo , Sequência Conservada , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Expressão Gênica , Células HeLa , Humanos , Mutação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Imagem com Lapso de TempoRESUMO
BACKGROUND: Improving cancer immunotherapy long-term clinical benefit is a major priority. It has become apparent that multiple axes of immune suppression restrain the capacity of T cells to provide anti-tumour activity including signalling through PD1/PD-L1 and LAG3/MHC-II. METHODS: CB213 has been developed as a fully human PD1/LAG3 co-targeting multi-specific Humabody composed of linked VH domains that avidly bind and block PD1 and LAG3 on dual-positive T cells. We present the preclinical primary pharmacology of CB213: biochemistry, cell-based function vs. immune-suppressive targets, induction of T cell proliferation ex vivo using blood obtained from NSCLC patients, and syngeneic mouse model anti-tumour activity. CB213 pharmacokinetics was assessed in cynomolgus macaques. RESULTS: CB213 shows picomolar avidity when simultaneously engaging PD1 and LAG3. Assessing LAG3/MHC-II or PD1/PD-L1 suppression individually, CB213 preferentially counters the LAG3 axis. CB213 showed superior activity vs. αPD1 antibody to induce ex vivo NSCLC patient T cell proliferation and to suppress tumour growth in a syngeneic mouse tumour model, for which both experimental systems possess PD1 and LAG3 suppressive components. Non-human primate PK of CB213 suggests weekly clinical administration. CONCLUSIONS: CB213 is poised to enter clinical development and, through intercepting both PD1 and LAG3 resistance mechanisms, may benefit patients with tumours escaping front-line immunological control.
Assuntos
Antígenos CD/imunologia , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Animais , Antígenos CD/metabolismo , Antígeno B7-H1 , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Receptor de Morte Celular Programada 1 , Linfócitos T , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
The Anaphase Promoting Complex/Cyclosome (APC/C) in complex with its co-activator Cdc20 is responsible for targeting proteins for ubiquitin-mediated degradation during mitosis. The activity of APC/C-Cdc20 is inhibited during prometaphase by the Spindle Assembly Checkpoint (SAC) yet certain substrates escape this inhibition. Nek2A degradation during prometaphase depends on direct binding of Nek2A to the APC/C via a C-terminal MR dipeptide but whether this motif alone is sufficient is not clear. Here, we identify Kif18A as a novel APC/C-Cdc20 substrate and show that Kif18A degradation depends on a C-terminal LR motif. However in contrast to Nek2A, Kif18A is not degraded until anaphase showing that additional mechanisms contribute to Nek2A degradation. We find that dimerization via the leucine zipper, in combination with the MR motif, is required for stable Nek2A binding to and ubiquitination by the APC/C. Nek2A and the mitotic checkpoint complex (MCC) have an overlap in APC/C subunit requirements for binding and we propose that Nek2A binds with high affinity to apo-APC/C and is degraded by the pool of Cdc20 that avoids inhibition by the SAC.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Cinesinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Complexos Ubiquitina-Proteína Ligase/fisiologia , Ciclossomo-Complexo Promotor de Anáfase , Subunidade Apc8 do Ciclossomo-Complexo Promotor de Anáfase , Proteínas Cdc20 , Proteínas de Ciclo Celular/metabolismo , Células HeLa , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Quinases Relacionadas a NIMA , Prometáfase/fisiologia , Ligação Proteica , Multimerização Proteica , Fatores de Tempo , Células Tumorais Cultivadas , Complexos Ubiquitina-Proteína Ligase/metabolismoRESUMO
Ubiquitin-mediated proteolysis is critical for the alternation between DNA replication and mitosis and for the key regulatory events in mitosis. The anaphase-promoting complex/cyclosome (APC/C) is a conserved ubiquitin ligase that has a fundamental role in regulating mitosis and the cell cycle in all eukaryotes. In vertebrate cells, early mitotic inhibitor 1 (Emi1) has been proposed as an important APC/C inhibitor whose destruction may trigger activation of the APC/C at mitosis. However, in this study, we show that the degradation of Emi1 is not required to activate the APC/C in mitosis. Instead, we uncover a key role for Emi1 in inhibiting the APC/C in interphase to stabilize the mitotic cyclins and geminin to promote mitosis and prevent rereplication. Thus, Emi1 plays a crucial role in the cell cycle to couple DNA replication with mitosis, and our results also question the current view that the APC/C has to be inactivated to allow DNA replication.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA/fisiologia , Proteínas F-Box/metabolismo , Interfase/fisiologia , Mitose/fisiologia , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Ciclinas/metabolismo , Geminina , Células HeLa , HumanosRESUMO
The GTPase Ran regulates nucleocytoplasmic transport in interphase and spindle organisation in mitosis via effectors of the importin beta superfamily. Ran-binding protein 1 (RanBP1) regulates guanine nucleotide turnover on Ran, as well as its interactions with effectors. Unlike other Ran network members that are steadily expressed, RanBP1 abundance is modulated during the mammalian cell cycle, peaking in mitosis and declining at mitotic exit. Here, we show that RanBP1 downregulation takes place in mid to late telophase, concomitant with the reformation of nuclei. Mild RanBP1 overexpression in murine cells causes RanBP1 to persist in late mitosis and hinders a set of events underlying the telophase to interphase transition, including chromatin decondensation, nuclear expansion and nuclear lamina reorganisation. Moreover, the reorganisation of nuclear pores fails associated with defective nuclear relocalisation of NLS cargoes. Co-expression of importin beta, together with RanBP1, however mitigates these defects. Thus, RanBP1 downregulation is required for nuclear reorganisation pathways operated by importin beta after mitosis.
Assuntos
Núcleo Celular/enzimologia , Regulação para Baixo , Mitose , Proteínas Nucleares/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Proteínas Nucleares/genética , Proteína ran de Ligação ao GTP/genéticaRESUMO
BACKGROUND: The mitotic kinases, Cdk1, Aurora A/B, and Polo-like kinase 1 (Plk1) have been characterized extensively to further understanding of mitotic mechanisms and as potential targets for cancer therapy. Cdk1 and Aurora kinase studies have been facilitated by small-molecule inhibitors, but few if any potent Plk1 inhibitors have been identified. RESULTS: We describe the cellular effects of a novel compound, BI 2536, a potent and selective inhibitor of Plk1. The fact that BI 2536 blocks Plk1 activity fully and instantaneously enabled us to study controversial and unknown functions of Plk1. Cells treated with BI 2536 are delayed in prophase but eventually import Cdk1-cyclin B into the nucleus, enter prometaphase, and degrade cyclin A, although BI 2536 prevents degradation of the APC/C inhibitor Emi1. BI 2536-treated cells lack prophase microtubule asters and thus polymerize mitotic microtubules only after nuclear-envelope breakdown and form monopolar spindles that do not stably attach to kinetochores. Mad2 accumulates at kinetochores, and cells arrest with an activated spindle-assembly checkpoint. BI 2536 prevents Plk1's enrichment at kinetochores and centrosomes, and when added to metaphase cells, it induces detachment of microtubules from kinetochores and leads to spindle collapse. CONCLUSIONS: Our results suggest that Plk1's accumulation at centrosomes and kinetochores depends on its own activity and that this activity is required for maintaining centrosome and kinetochore function. Our data also show that Plk1 is not required for prophase entry, but delays transition to prometaphase, and that Emi1 destruction in prometaphase is not essential for APC/C-mediated cyclin A degradation.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas F-Box/metabolismo , Microtúbulos/efeitos dos fármacos , Mitose/fisiologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Pteridinas/farmacologia , Fuso Acromático/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/metabolismo , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Pteridinas/metabolismo , Fuso Acromático/metabolismo , Quinase 1 Polo-LikeRESUMO
The targeted destruction of key regulators helps to drive the cell cycle. Here we describe a quantitative assay to measure destruction of different regulators in mitotic cells. This assay uses GFP-tagged substrates and time-lapse fluorescence microscopy of single cells to pinpoint the timing of destruction of different substrates at different stages in mitosis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Mitose/fisiologia , Fuso Acromático/fisiologia , Anáfase/fisiologia , Cromossomos Humanos/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Metáfase/fisiologia , Microinjeções , Microscopia de Fluorescência , RNA Mensageiro/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The small Ran GTPase, a key regulator of nucleocytoplasmic transport, is also involved in microtubule assembly and nuclear membrane formation. Herein, we show by immunofluorescence, immunoelectron microscopy, and biochemical analysis that a fraction of Ran is tightly associated with the centrosome throughout the cell cycle. Ran interaction with the centrosome is mediated by the centrosomal matrix A kinase anchoring protein (AKAP450). Accordingly, when AKAP450 is delocalized from the centrosome, Ran is also delocalized, and as a consequence, microtubule regrowth or anchoring is altered, despite the persisting association of gamma-tubulin with the centrosome. Moreover, Ran is recruited to Xenopus sperm centrosome during its activation for microtubule nucleation. We also demonstrate that centrosomal proteins such as centrin and pericentrin, but not gamma-tubulin, AKAP450, or ninein, undertake a nucleocytoplasmic exchange as they concentrate in the nucleus upon export inhibition by leptomycin B. Together, these results suggest a challenging possibility, namely, that centrosome activity could depend upon nucleocytoplasmic exchange of centrosomal proteins and local Ran-dependent concentration at the centrosome.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Ciclo Celular/fisiologia , Centrossomo/metabolismo , Proteínas do Citoesqueleto , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Proteínas de Ancoragem à Quinase A , Antígenos/metabolismo , Fracionamento Celular , Clonagem Molecular , Ácidos Graxos Insaturados/farmacologia , Imunofluorescência , Proteínas de Ligação ao GTP/metabolismo , Células HeLa , Humanos , Microscopia Imunoeletrônica , Modelos Moleculares , Transporte Proteico , Combinação Trimetoprima e Sulfametoxazol/metabolismo , Tubulina (Proteína)/metabolismo , Células Tumorais CultivadasRESUMO
The Ran GTPase has important roles in nucleocytoplasmic transport, cell cycle progression, nuclear organization and nuclear envelope (NE) assembly. In this review, we discuss emerging evidence that implicate the Ran GTPase system in mitotic control in mammalian cells. Recent work indicates that members of the Ran network control two fundamental aspects of the mammalian mitotic apparatus: (i) centrosome and spindle pole function, and (ii) kinetochore function. It is also emerging that, after NE breakdown, specific Ran network components assemble in local combinations at crucial sites of the mitotic apparatus. In the light of these findings, the original notion that nucleotide-bound forms of the Ran GTPase are distributed along a unique "gradient" in mitotic cells should be re-examined. Available data also suggest that the Ran system is deregulated in certain cellular contexts: this may represent a favoring condition for the onset and propagation of mitotic errors that can predispose cells to become genetically unstable and facilitate neoplastic growth.
Assuntos
Mitose , Proteína ran de Ligação ao GTP/metabolismo , Animais , Centrossomo/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Fuso Acromático/metabolismoRESUMO
The anaphase-promoting complex or cyclosome (APC/C) is the ubiquitin ligase that regulates mitosis by targeting specific proteins for degradation at specific times under the control of the spindle assembly checkpoint (SAC). How the APC/C recognizes its different substrates is a key problem in the control of cell division. Here, we have identified the ABBA motif in cyclin A, BUBR1, BUB1, and Acm1, and we show that it binds to the APC/C coactivator CDC20. The ABBA motif in cyclin A is required for its proper degradation in prometaphase through competing with BUBR1 for the same site on CDC20. Moreover, the ABBA motifs in BUBR1 and BUB1 are necessary for the SAC to work at full strength and to recruit CDC20 to kinetochores. Thus, we have identified a conserved motif integral to the proper control of mitosis that connects APC/C substrate recognition with the SAC.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas Cdc20/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Mitose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclo Celular/fisiologia , Humanos , Cinetocoros/metabolismo , Estrutura Terciária de ProteínaRESUMO
The anaphase-promoting complex/cyclosome (APC/C) is the ubiquitin ligase essential to mitosis, which ensures that specific proteins are degraded at specific times to control the order of mitotic events. The APC/C coactivator, Cdc20, is targeted by the spindle assembly checkpoint (SAC) to restrict APC/C activity until metaphase, yet early substrates, such as cyclin A, are degraded in the presence of the active checkpoint. Cdc20 and the cyclin-dependent kinase cofactor, Cks, are required for cyclin A destruction, but how they enable checkpoint-resistant destruction has not been elucidated. In this study, we answer this problem: we show that the N terminus of cyclin A binds directly to Cdc20 and with sufficient affinity that it can outcompete the SAC proteins. Subsequently, the Cks protein is necessary and sufficient to promote cyclin A degradation in the presence of an active checkpoint by binding cyclin A-Cdc20 to the APC/C.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiologia , Ciclina A/metabolismo , Fuso Acromático/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Animais , Quinases relacionadas a CDC2 e CDC28 , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas Cdc20 , Proteínas de Ciclo Celular/genética , Ciclina A/genética , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Células HeLa , Humanos , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , UbiquitinaçãoRESUMO
Ordered progression through the cell cycle is essential to maintain genomic stability, and fundamental to this is ubiquitin-mediated proteolysis. In particular, the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase destabilises specific regulators at defined times in the cycle to ensure that each round of DNA replication is followed by cell division. Thus, the proper regulation of the APC/C is crucial in each cell cycle. There are several APC/C regulators that restrict its activity to specific cell cycle phases, and amongst these the early mitotic inhibitor 1 (Emi1) protein has recently come to prominence. Emi1 has been proposed to control APC/C in early mitosis; however, recent evidence questions this role. In this review we discuss new evidence that indicates that Emi1 is essential to restrict APC/C activity in interphase and, by doing so, ensure the proper coordination between DNA replication and mitosis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos/fisiologia , Replicação do DNA/fisiologia , Proteínas F-Box/metabolismo , Mitose/fisiologia , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Transdução de Sinais/fisiologiaRESUMO
The Ran GTPase plays a central function in control of nucleo-cytoplasmic transport in interphase. Mitotic roles of Ran have also been firmly established in Xenopus oocyte extracts. In this system, Ran-GTP, or the RCC1 exchange factor for Ran, drive spindle assembly by regulating the availability of 'aster-promoting activities'. In previous studies to assess whether the Ran network also influences mitosis in mammalian cells, we found that overexpression of Ran-binding protein 1 (RanBP1), a major effector of Ran, induces multipolar spindles. We now show that these abnormal spindles are generated through loss of cohesion in mitotic centrosomes. Specifically, RanBP1 excess induces splitting of mother and daughter centrioles at spindle poles; the resulting split centrioles can individually organize functional microtubule arrays, giving rise to functional spindle poles. RanBP1-dependent centrosome splitting is specifically induced in mitosis and requires microtubule integrity and Eg5 activity. In addition, we have identified a fraction of RanBP1 at the centrosome. These data indicate that overexpressed RanBP1 interferes with crucial factor(s) that control structural and dynamic features of centrosomes during mitosis and contribute to uncover novel mitotic functions downstream of the Ran network.
Assuntos
Centrossomo/metabolismo , Cinesinas/metabolismo , Mitose/fisiologia , Proteínas Nucleares/metabolismo , Fuso Acromático/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Núcleo Celular/metabolismo , Cromossomos/metabolismo , Feminino , Células HeLa , Humanos , Interfase , Camundongos , Microscopia de Fluorescência , Microtúbulos/metabolismo , Células NIH 3T3RESUMO
Spatial control is a key issue in cell division. The Ran GTPase regulates several fundamental processes for cell life, largely acting through importin molecules. The best understood of these is protein import through the nuclear envelope in interphase, but roles in mitotic spindle assembly are also established. In mammalian cells, in which centrosomes are major spindle organizers, a link is emerging between the Ran network, centrosomes and spindle poles. Here, we show that, after nuclear envelope breakdown, importin beta is transported to the spindle poles in mammalian cells. This localization is temporally regulated from prometaphase until anaphase, when importin beta dissociates from poles and is recruited back around reforming nuclei. Importin beta sediments with mitotic microtubules in vitro and its accumulation at poles requires microtubule integrity and dynamics in vivo. Furthermore, RNA interference-dependent inactivation of TPX2, the major Ran-dependent spindle organizer, abolishes importin beta accumulation at poles. Importin beta has a functional role in spindle pole organization, because overexpression yields mitotic spindles with abnormal, fragmented poles. Coexpression of TPX2 with importin beta mitigates these abnormalities. Together, these results indicate that the balance between importins and spindle regulators of the TPX2 type is crucial for spindle formation. Targeting of TPX2/importin-beta complexes to poles is a key aspect in Ran-dependent control of the mitotic apparatus in mammalian cells.