RESUMEN
Recent results indicate that regulating the balance between cyclin-B-Cdc2 kinase, also known as M-phase-promoting factor (MPF), and protein phosphatase 2A (PP2A) is crucial to enable correct mitotic entry and exit. In this work, we studied the regulatory mechanisms controlling the cyclin-B-Cdc2 and PP2A balance by analysing the activity of the Greatwall kinase and PP2A, and the different components of the MPF amplification loop (Myt1, Wee1, Cdc25) during the first embryonic cell cycle. Previous data indicated that the Myt1-Wee1-Cdc25 equilibrium is tightly regulated at the G2-M and M-G1 phase transitions; however, no data exist regarding the regulation of this balance during M phase and interphase. Here, we demonstrate that constant regulation of the cyclin-B-Cdc2 amplification loop is required for correct mitotic division and to promote correct timing of mitotic entry. Our results show that removal of Cdc25 from metaphase-II-arrested oocytes promotes mitotic exit, whereas depletion of either Myt1 or Wee1 in interphase egg extracts induces premature mitotic entry. We also provide evidence that, besides the cyclin-B-Cdc2 amplification loop, the Greatwall-PP2A pathway must also be tightly regulated to promote correct first embryonic cell division. When PP2A is prematurely inhibited in the absence of cyclin-B-Cdc2 activation, endogenous cyclin-A-Cdc2 activity induces irreversible aberrant mitosis in which there is, first, partial transient phosphorylation of mitotic substrates and, second, subsequent rapid and complete degradation of cyclin A and cyclin B, thus promoting premature and rapid exit from mitosis.
Asunto(s)
Embrión no Mamífero/citología , Embrión no Mamífero/fisiología , Factor Promotor de Maduración/metabolismo , Metafase/fisiología , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/fisiología , Proteínas de Xenopus/metabolismo , Animales , Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclina A/metabolismo , Ciclina B/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Xenopus laevis , Fosfatasas cdc25/metabolismoRESUMEN
XENOPUS: egg extracts are a powerful in vitro tool for studying complex biological processes, including nuclear reconstitution, nuclear membrane and pore assembly, and spindle assembly. Extracts have been further used to demonstrate a moonlighting regulatory role for nuclear import receptors or importins on these cell cycle assembly events. Here we show that exportins can also play a role in these events. Addition of Crm1, Exportin-t, or Exportin-5 decreased nuclear pore assembly in vitro. RanQ69L-GTP, a constitutively active form of RanGTP, ameliorated inhibition. Both Crm1 and Exportin-t inhibited fusion of nuclear membranes, again counteracted by RanQ69L-GTP. In mitotic extracts, Crm1 and Exportin-t negatively impacted spindle assembly. Pulldowns from the extracts using Crm1- or Exportin-t-beads revealed nucleoporins known to be essential for both nuclear pore and spindle assembly, with RanQ69L-GTP decreasing a subset of these target interactions. This study suggests a model where exportins, like importins, can regulate major mitotic assembly events.
Asunto(s)
Carioferinas/metabolismo , Fusión de Membrana , Mitosis , Poro Nuclear/metabolismo , Huso Acromático/metabolismo , Animales , Sistema Libre de Células/metabolismo , Humanos , Carioferinas/genética , Poro Nuclear/genética , Huso Acromático/genética , Xenopus laevisRESUMEN
During mitosis, the Xenopus chromokinesin Kid (Xkid) provides the polar ejection forces needed at metaphase for chromosome congression, and its degradation is required at anaphase to induce chromosome segregation. Despite the fact that the degradation of Xkid at anaphase seems to be a key regulatory factor to induce chromosome movement to the poles, little is known about the mechanisms controlling this proteolysis. We investigated here the degradation pathway of Xkid. We demonstrate that Xkid is degraded both in vitro and in vivo by APC/Cdc20 and APC/Cdh1. We show that, despite the presence of five putative D-box motifs in its sequence, Xkid is proteolyzed in a D-box-independent manner. We identify a domain within the C terminus of this chromokinesin, with sequence GxEN, whose mutation completely stabilizes this protein by both APC/Cdc20 and APC/Cdh1. Moreover, we show that this degradation sequence acts as a transposable motif and induces the proteolysis of a GST-GXEN fusion protein. Finally, we demonstrate that both a D-box and a GXEN-containing peptides completely block APC-dependent degradation of cyclin B and Xkid, indicating that the GXEN domain might mediate the recognition and association of Xkid with the APC.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Cinesinas/metabolismo , Secuencias de Aminoácidos , Anafase , Animales , Western Blotting , ADN Complementario/metabolismo , Proteínas de Unión al ADN/genética , Glutatión Transferasa/metabolismo , Metafase , Mitosis , Modelos Genéticos , Mutagénesis Sitio-Dirigida , Mutación , Oocitos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , Xenopus , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMEN
The spindle checkpoint prevents anaphase onset until all the chromosomes have successfully attached to the spindle microtubules. The mechanisms by which unattached kinetochores trigger and transmit a primary signal are poorly understood, although it seems to be dependent at least in part, on the kinetochore localization of the different checkpoint components. By using protein immunodepletion and mRNA translation in Xenopus egg extracts, we have studied the hierarchic sequence and the interdependent network that governs protein recruitment at the kinetochore in the spindle checkpoint pathway. Our results show that the first regulatory step of this cascade is defined by Aurora B/INCENP complex. Aurora B/INCENP controls the activation of a second regulatory level by inducing at the kinetochore the localization of Mps1, Bub1, Bub3, and CENP-E. This localization, in turn, promotes the recruitment to the kinetochore of Mad1/Mad2, Cdc20, and the anaphase promoting complex (APC). Unlike Aurora B/INCENP, Mps1, Bub1, and CENP-E, the downstream checkpoint protein Mad1 does not regulate the kinetochore localization of either Cdc20 or APC. Similarly, Cdc20 and APC do not require each other to be localized at these chromosome structures. Thus, at the last step of the spindle checkpoint cascade, Mad1/Mad2, Cdc20, and APC are recruited at the kinetochores independently from each other.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cinetocoros/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Huso Acromático/metabolismo , Animales , Aurora Quinasas , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Oocitos/citología , Oocitos/fisiología , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/fisiología , Huso Acromático/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMEN
Events controlling cell division are governed by the degradation of different regulatory proteins by the ubiquitin-dependent pathway. In this pathway, the attachment of a polyubiquitin chain to a substrate by an ubiquitin-ligase targets this substrate for degradation by the 26S proteasome. Two different ubiquitin ligases play an important role in the cell cycle: the SCF (Skp1/Cullin/F-box) and the anaphase-promoting complex (APC). In this review, we describe the present knowledge about the APC. We pay particular attention to the latest results concerning APC structure, APC regulation and substrate recognition, and we discuss the implication of these findings in the understanding the APC function.
Asunto(s)
Anafase/fisiología , Ciclo Celular/fisiología , Enzimas Ubiquitina-Conjugadoras/fisiología , Complejos de Ubiquitina-Proteína Ligasa/fisiología , Ciclosoma-Complejo Promotor de la Anafase , Animales , División Celular , Replicación del ADN , Homeostasis , Mitosis/fisiología , Ubiquitina/metabolismoRESUMEN
Events controlling cell division are governed by the degradation of different regulatory proteins by the ubiquitin-dependent pathway. In this pathway, the attachment of a polyubiquitin chain to a substrate by an ubiquitin-ligase targets this substrate for degradation. Xenopus egg extracts present many advantages for the study of the cell cycle, including the availability of a large quantity of material synchronized at a particular phase of the cell cycle. In this chapter, we describe various protocols used in Xenopus egg extracts to study the ubiquitination and degradation of different cell cycle regulators. We first provide the method used to obtain interphase- and metaphase II-arrested egg extracts. Subsequently, we describe the protocol employed in these extracts to test the putative ubiquitination and degradation of a protein. Moreover, we describe a detailed practical procedure to test the role of different regulators in the ubiquitin-dependent degradation pathway of a specific protein. To that, we show how to eliminate some of these regulators from the extracts by immunodepletion and how to activate ectopically their function by the translation of their messenger ribonucleic acid. Finally, the Notes provide a series of practical details that explain the different problems that can occur and the possible solutions used to overcome them.
Asunto(s)
Óvulo/metabolismo , Proteínas/metabolismo , Ubiquitina/química , Xenopus , Animales , Extractos Celulares/química , Óvulo/química , Proteínas/química , Ubiquitina/fisiologíaRESUMEN
The unexpected repurposing of nuclear transport proteins from their function in interphase to an equally vital and very different set of functions in mitosis was very surprising. The multi-talented cast when first revealed included the import receptors, importin alpha and beta, the small regulatory GTPase RanGTP, and a subset of nuclear pore proteins. In this review, we report that recent years have revealed new discoveries in each area of this expanding story in vertebrates: (a) The cast of nuclear import receptors playing a role in mitotic spindle regulation has expanded: both transportin, a nuclear import receptor, and Crm1/Xpo1, an export receptor, are involved in different aspects of spindle assembly. Importin beta and transportin also regulate nuclear envelope and pore assembly. (b) The role of nucleoporins has grown to include recruiting the key microtubule nucleator - the γ-TuRC complex - and the exportin Crm1 to the mitotic kinetochores of humans. Together they nucleate microtubule formation from the kinetochores toward the centrosomes. (c) New research finds that the original importin beta/RanGTP team have been further co-opted by evolution to help regulate other cellular and organismal activities, ranging from the actual positioning of the spindle within the cell perimeter, to regulation of a newly discovered spindle microtubule branching activity, to regulation of the interaction of microtubule structures with specific actin structures. (d) Lastly, because of the multitudinous roles of karyopherins throughout the cell cycle, a recent large push toward testing their potential as chemotherapeutic targets has begun to yield burgeoning progress in the clinic.
Asunto(s)
Mitosis , Actinas/metabolismo , Transporte Activo de Núcleo Celular , Animales , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Humanos , Proteína de Unión al GTP ran/metabolismoRESUMEN
The unexpected repurposing of nuclear transport proteins from their function in interphase to an equally vital and very different set of functions in mitosis was very surprising. The multi-talented cast when first revealed included the import receptors, importin alpha and beta, the small regulatory GTPase RanGTP, and a subset of nuclear pore proteins. In this review, we report that recent years have revealed new discoveries in each area of this expanding story in vertebrates: (a) The cast of nuclear import receptors playing a role in mitotic spindle regulation has expanded: both transportin, a nuclear import receptor, and Crm1/Xpo1, an export receptor, are involved in different aspects of spindle assembly. Importin beta and transportin also regulate nuclear envelope and pore assembly. (b) The role of nucleoporins has grown to include recruiting the key microtubule nucleator the γ-TuRC complex and the exportin Crm1 to the mitotic kinetochores of humans. Together they nucleate microtubule formation from the kinetochores toward the centrosomes. (c) New research finds that the original importin beta/RanGTP team have been further co-opted by evolution to help regulate other cellular and organismal activities, ranging from the actual positioning of the spindle within the cell perimeter, to regulation of a newly discovered spindle microtubule branching activity, to regulation of the interaction of microtubule structures with specific actin structures. (d) Lastly, because of the multitudinous roles of karyopherins throughout the cell cycle, a recent large push toward testing their potential as chemotherapeutic targets has begun to yield burgeoning progress in the clinic.
RESUMEN
The large and complex eukaryotic nucleus is the arbiter of DNA replication, RNA transcription, splicing, and ribosome assembly. With the advent of in vitro nuclear reconstitution extracts derived from Xenopus eggs in the 1980s, it became possible to assemble multiple nuclei in vitro around added DNA or chromatin substrates. Such reconstituted nuclei contain a nuclear lamina, double nuclear membranes, nuclear pores, and are competent for DNA replication and nuclear import. In vitro nuclear reconstitution has allowed the assembly of "wild-type" and "biochemically mutant" nuclei in which the impact of individual components can be assessed. Here, we describe protocols for preparation of the nuclear reconstitution extract, nuclear reconstitution in vitro, assessment of nuclear membrane integrity, and a more specialized assay for nuclear pore assembly into preformed pore-free nuclear intermediates.
Asunto(s)
Núcleo Celular/metabolismo , Poro Nuclear/metabolismo , Óvulo/citología , Animales , Cromatina/genética , ADN/genética , Replicación del ADN , Proteínas Nucleares , Xenopus laevisRESUMEN
The nuclear import receptors importin ß and transportin play a different role in mitosis: both act phenotypically as spatial regulators to ensure that mitotic spindle, nuclear membrane, and nuclear pore assembly occur exclusively around chromatin. Importin ß is known to act by repressing assembly factors in regions distant from chromatin, whereas RanGTP produced on chromatin frees factors from importin ß for localized assembly. The mechanism of transportin regulation was unknown. Diametrically opposed models for transportin action are as follows: 1) indirect action by RanGTP sequestration, thus down-regulating release of assembly factors from importin ß, and 2) direct action by transportin binding and inhibiting assembly factors. Experiments in Xenopus assembly extracts with M9M, a superaffinity nuclear localization sequence that displaces cargoes bound by transportin, or TLB, a mutant transportin that can bind cargo and RanGTP simultaneously, support direct inhibition. Consistently, simple addition of M9M to mitotic cytosol induces microtubule aster assembly. ELYS and the nucleoporin 107-160 complex, components of mitotic kinetochores and nuclear pores, are blocked from binding to kinetochores in vitro by transportin, a block reversible by M9M. In vivo, 30% of M9M-transfected cells have spindle/cytokinesis defects. We conclude that the cell contains importin ß and transportin "global positioning system"or "GPS" pathways that are mechanistically parallel.
Asunto(s)
Proteínas de Drosophila/metabolismo , Carioferinas/metabolismo , Mitosis , Proteínas de Xenopus/metabolismo , Proteína de Unión al GTP ran/metabolismo , Secuencia de Aminoácidos , Animales , Extractos Celulares , Cromatina/metabolismo , Citocinesis , Proteínas de Drosophila/química , Células HeLa , Humanos , Interfase , Carioferinas/química , Cinetocoros/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Membrana Nuclear/metabolismo , Señales de Localización Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Huso Acromático/metabolismo , Xenopus , beta Carioferinas/metabolismoRESUMEN
Mitosis in higher eukaryotes is marked by the sequential assembly of two massive structures: the mitotic spindle and the nucleus. Nuclear assembly itself requires the precise formation of both nuclear membranes and nuclear pore complexes. Previously, importin alpha/beta and RanGTP were shown to act as dueling regulators to ensure that these assembly processes occur only in the vicinity of the mitotic chromosomes. We now find that the distantly related karyopherin, transportin, negatively regulates nuclear envelope fusion and nuclear pore assembly in Xenopus egg extracts. We show that transportin-and importin beta-initiate their regulation as early as the first known step of nuclear pore assembly: recruitment of the critical pore-targeting nucleoporin ELYS/MEL-28 to chromatin. Indeed, each karyopherin can interact directly with ELYS. We further define the nucleoporin subunit targets for transportin and importin beta and find them to be largely the same: ELYS, the Nup107/160 complex, Nup53, and the FG nucleoporins. Equally importantly, we find that transportin negatively regulates mitotic spindle assembly. These negative regulatory events are counteracted by RanGTP. We conclude that the interplay of the two negative regulators, transportin and importin beta, along with the positive regulator RanGTP, allows precise choreography of multiple cell cycle assembly events.
Asunto(s)
Carioferinas/metabolismo , Mitosis , Poro Nuclear/metabolismo , Huso Acromático/metabolismo , Secuencia de Aminoácidos , Animales , Cromatina/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Humanos , Fusión de Membrana , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas de Complejo Poro Nuclear/metabolismo , Unión Proteica , Transporte de Proteínas , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Xenopus , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , beta Carioferinas/metabolismo , Proteína de Unión al GTP ran/metabolismoRESUMEN
The anaphase-promoting complex (APC) early mitotic inhibitor 1 (Emi1) is required to induce S- and M-phase entries by stimulating the accumulation of cyclin A and cyclin B through APC(Cdh1/cdc20) inhibition. In this report, we show that Emi1 proteolysis can be induced by cyclin A/cdk (cdk for cyclin-dependent kinase). Paradoxically, Emi1 is stable during G2 phase, when cyclin A/cdk, Plx1 and SCF(betatrcp) (SCF for Skp1-Cul1-Fbox protein)--which play a role in its degradation--are active. Here, we identify Pin1 as a new regulator of Emi1 that induces Emi1 stabilization by preventing its association with SCF(betatrcp). We show that Pin1 binds to Emi1 and prevents its association with betatrcp in an isomerization-dependent pathway. We also show that Emi1-Pin1 binding is present in vivo in XL2 cells during G2 phase and that this association protects Emi1 from being degraded during this phase of the cell cycle. We propose that S- and M-phase entries are mediated by the accumulation of cyclin A and cyclin B through a Pin1-dependent stabilization of Emi1 during G2.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Isomerasa de Peptidilprolil/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas con Repetición de beta-Transducina/metabolismo , Animales , Proteínas de Ciclo Celular/análisis , Proteínas de Ciclo Celular/genética , Extractos Celulares/química , Ciclina A/metabolismo , Ciclina A/farmacología , Ciclina B/metabolismo , Ciclina B/farmacología , Quinasas Ciclina-Dependientes/farmacología , Fase G2 , Humanos , Inmunoprecipitación , Mitosis , Peptidilprolil Isomerasa de Interacción con NIMA , Xenopus , Proteínas de Xenopus/análisis , Proteínas de Xenopus/genéticaRESUMEN
Neuronal apoptosis has been shown to require de novo RNA/protein synthesis. However, very few genes whose expression is necessary for inducing apoptosis have been identified so far. To systematically identify such genes, we have used genome-scale, long oligonucleotide microarrays and characterized the gene expression profile of cerebellar granule neurons in the early phase of apoptosis elicited by KCl deprivation. We identified 368 significantly differentially expressed genes, including most of the genes previously reported to be transcriptionally regulated in this paradigm. In addition, we identified several hundreds of genes whose transcriptional regulation has never been associated with neuronal apoptosis. We used automated Gene Ontology annotation, analysis of promoter sequences, and statistical tools to characterize these regulations. Although differentially expressed genes included some components of the apoptotic machinery, this functional category was not significantly over-represented among regulated genes. On the other hand, categories related to signal transduction were the most significantly over-represented group. This indicates that the apoptotic machinery is mainly constitutive, whereas molecular pathways that lead to the activation of apoptotic components are transcriptionally regulated. In particular, we show for the first time that signaling pathways known to be involved in the control of neuronal survival are regulated at the transcriptional level and not only by post-translational mechanisms. Moreover, our approach provides insights into novel transcription factors and novel mechanisms, such as the unfolded protein response and cell adhesion, that may contribute to the induction of neuronal apoptosis.
Asunto(s)
Apoptosis/genética , Perfilación de la Expresión Génica , Neuronas/metabolismo , Transducción de Señal/genética , Transcripción Genética/genética , Animales , Supervivencia Celular , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Neuronas/citología , Neuronas/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Cloruro de Potasio/farmacología , Regiones Promotoras Genéticas/genética , Pliegue de Proteína , ARN Mensajero/análisis , ARN Mensajero/genética , Elementos de Respuesta/genética , Factores de Tiempo , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
We have demonstrated previously that Xenopus Aurora-A is degraded at late mitosis by the APC/Fizzy-Related in a D-Box-dependent manner. Here we demonstrate that, although Aurora-B possesses the same D-Box as Aurora-A, Aurora-B is not degraded by this ubiquitin ligase. We have constructed a chimera Aurora-A/B with the N-terminus of Aurora-A and the C-terminus of Aurora-B and we have examined its degradation by APC/Fizzy-Related. We demonstrate that the N-terminus of Aurora-A confers degradation capacity on the C-terminus of Aurora-B and that this feature is blocked by mutation of the conserved D-Box sequence. We characterize the minimal degradation signal at the N-terminus of Aurora-A and demonstrate that its deletion blocks the degradation of this protein by APC/Fizzy-Related. Thus, we conclude that two different degradation signals are required for proteolysis of Aurora-A. The first one, which we designated D-Box-activating domain, within the N-terminal domain of Aurora-A confers the functionality to the second, a silent D-Box, present within the C-terminus of the kinase.