RESUMEN
We have studied the role of cyclins and cyclin-dependent kinase (CDK) activity in apoptosis induced by camptothecin (CPT). In this model, 22% of the cells stain for annexin-V at 24 h and then proceed to be 93% positive by 72 h. This time window permits the analysis of cyclins in cells that are committed to apoptosis but not yet dead. We provide evidence that cyclin protein levels and then associated kinase levels increase after CPT treatment. Strikingly, cyclin B1 and cyclin E1 proteins are present at the same time in CPT treated HT29 cells. Although cyclin B1 and E1 CDK complexes are activated in CPT treated cells, only the cyclin B1 complex is required for apoptosis since reduction of cyclin B1 by RNAi or roscovitine treatment reduces the number of annexin-V-stained cells. We have detected poorly organized chromosomes and phosphorylated histone H3 epitopes at the time of maximum cyclin B1/CDK kinase activity in CPT-treated cells, which suggests that these cells enter a mitotic catastrophe. Understanding which CDKs are required for apoptosis may allow us to better adapt CDK inhibitors for use as anti-cancer compounds.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Camptotecina/farmacología , Ciclina B/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Secuencia de Bases , Ciclo Celular , Ciclina A/metabolismo , Ciclina B1 , Ciclina E/metabolismo , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente , Células HT29 , Humanos , Interferencia de ARNRESUMEN
The actin cytoskeleton is a dynamic network that is composed of a variety of F-actin structures. To understand how these structures are produced, we tested the capacity of proteins to direct actin polymerization in a bead assay in vitro and in a mitochondrial-targeting assay in cells. We found that human zyxin and the related protein ActA of Listeria monocytogenes can generate new actin structures in a vasodilator-stimulated phosphoprotein-dependent (VASP) manner, but independently of the Arp2/3 complex. These results are consistent with the concept that there are multiple actin-polymerization machines in cells. With these simple tests it is possible to probe the specific function of proteins or identify novel molecules that act upon cellular actin polymerization.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas del Citoesqueleto , Proteínas de la Membrana/metabolismo , Metaloproteínas/metabolismo , Polímeros/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/ultraestructura , Proteína 2 Relacionada con la Actina , Proteína 3 Relacionada con la Actina , Animales , Bioensayo , Moléculas de Adhesión Celular/metabolismo , Sistema Libre de Células , Chlorocebus aethiops , Técnica del Anticuerpo Fluorescente , Glicoproteínas , Células HeLa/citología , Células HeLa/efectos de los fármacos , Células HeLa/metabolismo , Humanos , Metaloproteínas/genética , Proteínas de Microfilamentos , Microesferas , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Transfección , Células Vero/citología , Células Vero/efectos de los fármacos , Células Vero/metabolismo , Proteína del Síndrome de Wiskott-Aldrich , ZixinaAsunto(s)
Proteína Quinasa CDC2/metabolismo , Ciclo Celular/fisiología , Proteínas Quinasas/metabolismo , Fosfatasas cdc25/metabolismo , Animales , Proteína Quinasa CDC2/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Daño del ADN , Humanos , Neoplasias/fisiopatología , Proteínas Quinasas/genética , Fosfatasas cdc25/genéticaAsunto(s)
Comunicación , Encefalopatía Espongiforme Bovina/transmisión , Política , Investigación , Animales , Bovinos , HumanosRESUMEN
Zyxin contains a proline-rich N-terminal domain that is similar to the C-terminal domain in the ActA protein of the bacteria, Listeria monocytogenes. We screened the entire amino acid sequence of human zyxin for Mena-interacting peptides and found that, as with ActA, proline-rich sequences were the sole zyxin sequences capable of binding to Ena/vasodilator-stimulated phosphoprotein (VASP) family members in vitro. From this information, we tested zyxin mutants in which the proline-rich sequences were altered. The reduction in Mena/VASP binding was confirmed by peptide tests, immunoprecipitation, and ectopic expression of zyxin variants at the surface of mitochondria. By transfection assays we showed that zyxin interaction with Mena/VASP in vivo enhances the production of actin-rich structures at the apical surface of cells. Microinjection into cells of peptides corresponding to the first proline-rich sequence of zyxin caused the loss of Mena/VASP from focal contacts. Furthermore, these peptides reduced the degree of spreading of cells replated after trypsinization. We conclude that zyxin and proteins that harbor similar proline-rich repeats contribute to the positioning of Mena/VASP proteins. The positioning of Ena/VASP family members appears to be important when the actin cytoskeleton is reorganized, such as during spreading.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Metaloproteínas/metabolismo , Fosfoproteínas/metabolismo , Secuencia de Aminoácidos , Moléculas de Adhesión Celular/genética , Proteínas del Citoesqueleto , Glicoproteínas , Humanos , Listeria monocytogenes , Metaloproteínas/genética , Proteínas de Microfilamentos , Datos de Secuencia Molecular , Mutación , Fosfoproteínas/genética , Prolina , Unión Proteica , ZixinaRESUMEN
Inspired by the motility of the bacteria Listeria monocytogenes, we have experimentally studied the growth of an actin gel around spherical beads grafted with ActA, a protein known to be the promoter of bacteria movement. On ActA-grafted beads F-actin is formed in a spherical manner, whereas on the bacteria a "comet-like" tail of F-actin is produced. We show experimentally that the stationary thickness of the gel depends on the radius of the beads. Moreover, the actin gel is not formed if the ActA surface density is too low. To interpret our results, we propose a theoretical model to explain how the mechanical stress (due to spherical geometry) limits the growth of the actin gel. Our model also takes into account treadmilling of actin. We deduce from our work that the force exerted by the actin gel on the bacteria is of the order of 10 pN. Finally, we estimate from our theoretical model possible conditions for developing actin comet tails.
Asunto(s)
Actinas/química , Proteínas Bacterianas/química , Listeria monocytogenes/fisiología , Proteínas de la Membrana/química , Actinas/fisiología , Actinas/ultraestructura , Animales , Proteínas Bacterianas/fisiología , Proteínas Bacterianas/ultraestructura , Sistema Libre de Células , Citosol/fisiología , Geles , Glutatión Transferasa/química , Células HeLa , Humanos , Listeria monocytogenes/ultraestructura , Proteínas de la Membrana/fisiología , Proteínas de la Membrana/ultraestructura , Microscopía Electrónica , Microscopía Inmunoelectrónica , Modelos Biológicos , Movimiento , Músculo Esquelético , Conejos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/ultraestructura , Propiedades de SuperficieRESUMEN
The LPP gene is the preferred translocation partner of the HMGIC gene in a subclass of human benign mesenchymal tumors known as lipomas. Here we have characterized the LPP gene product that shares 41% of sequence identity with the focal adhesion protein zyxin. LPP localizes in focal adhesions as well as in cell-to-cell contacts, and it binds VASP, a protein implicated in the control of actin organization. In addition, LPP accumulates in the nucleus of cells upon treatment with leptomycin B, an inhibitor of the export factor CRM1. The nuclear export of LPP depends on an N-terminally located leucine-rich sequence that shares sequence homology with well-defined nuclear export signals. Moreover, LPP displays transcriptional activation capacity, as measured by GAL4-based assays. Altogether, these results show that the LPP protein has multifunctional domains and may serve as a scaffold upon which distinct protein complexes are assembled in the cytoplasm and in the nucleus.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Señales de Clasificación de Proteína , Activación Transcripcional , Células 3T3 , Secuencia de Aminoácidos , Animales , Fusión Artificial Génica , Sitios de Unión , Células CACO-2 , Moléculas de Adhesión Celular/metabolismo , Línea Celular Transformada , Núcleo Celular/metabolismo , Chlorocebus aethiops , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/inmunología , Ácidos Grasos Insaturados/farmacología , Expresión Génica , Glicoproteínas , Células HL-60 , Células HeLa , Proteínas del Grupo de Alta Movilidad/genética , Humanos , Proteínas con Dominio LIM , Células LLC-PK1 , Metaloproteínas/química , Ratones , Proteínas de Microfilamentos , Datos de Secuencia Molecular , Fosfoproteínas/metabolismo , Conejos , Porcinos , Células Vero , ZixinaRESUMEN
The concentration of proteins in cells is an important parameter that determines how a protein will interact with other proteins or pharmacological agents. Recent developments in Western blotting techniques have now made this a method of choice to measure protein concentration in complex solutions such as total cell extracts. We show that detection of Cy5-coupled secondary antibodies by PhosphorImager analysis produces signals that approach linearity with respect to protein concentration over a 20-fold range. We used this technique to estimate cellular levels of zyxin, which is an important protein component of the actin cytoskeleton in mammalian cells. By producing specific protein standards based on sequences that are available from public databases, it is now possible to estimate the concentration of almost any protein by this technique.
Asunto(s)
Western Blotting/métodos , Carbocianinas/química , Proteínas/análisis , Secuencia de Aminoácidos , Animales , Anticuerpos/química , Anticuerpos/inmunología , Calibración , Extractos Celulares/química , Línea Celular , Proteínas del Citoesqueleto , Glicoproteínas , Humanos , Metaloproteínas/análisis , Metaloproteínas/inmunología , Datos de Secuencia Molecular , ZixinaRESUMEN
The human protein kinase Plk1, a member of the polo-like kinase family, is known to function at mitosis. Here we show that the relative specific activity of Plk1 increases in mitosis, that Plk1 is specifically phosphorylated during mitosis, and that phosphatase treatment reduces mitotic Plk1 activity to interphase levels. To identify domains involved in the regulation of Plk1 activity, deletion mutants of Plk1 were constructed and their activities examined. Deletion of the extreme C-terminus of Plk1 substantially increased kinase activity, indicating that the C-terminus harbors an inhibitory domain. Finally, the consequences of over-production of wild-type and mutant Plk1 protein were analyzed, using transient transfection assays. Cells overexpressing Plk1 protein were able to enter mitosis and establish an apparently normal bipolar spindle. In contrast, progression through mitosis was transiently delayed, and cytokinesis appeared to be disturbed, as reflected by a significant increase in large cells with multiple, often fragmented nuclei. These results are relevant to recently proposed roles for Plks during both entry into and exit from mitosis.
Asunto(s)
Ciclo Celular , Proteínas Quinasas/metabolismo , Proteínas Quinasas/fisiología , Proteínas de Ciclo Celular , Activación Enzimática/genética , Fase G2 , Células HeLa , Humanos , Mitosis , Mutagénesis , Fosforilación , Proteínas Quinasas/biosíntesis , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas , Eliminación de Secuencia , Quinasa Tipo Polo 1RESUMEN
The intracellular bacterial parasite Listeria monocytogenes produces ActA protein at its surface to facilitate the localized assembly of actin-filled comets that are required for movement. The organization of actin in Listeria comets shows striking similarity to the organization of actin at the plasma membrane of mammalian cells. Therefore we examined the possibility that an ActA-like protein is present in mammalian cells. By using antibodies directed against ActA, we identified zyxin as an ActA related protein in a number of cell types. We compared the functions of ActA and zyxin by transient expression of variants tagged with an inner plasma membrane localization sequence (a CAAX box). Targeting of the proline rich domain of zyxin to the plasma membrane disrupts the actin cytoskeleton and cell shape in a manner similar to that which occurs with membrane-targeted ActA sequences. A chimeric protein composed of the N-terminal domain of ActA fused to the N-terminal and central domains of zyxin induced a full ActA response in cells. Furthermore, zyxin and ActA exhibit common protein partners in vitro. On the basis of the shared properties of zyxin and ActA, we propose that zyxin enhances actin organizing activity in mammalian cells.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Listeria monocytogenes/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Metaloproteínas/química , Metaloproteínas/metabolismo , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos , Secuencia de Bases , Línea Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Chlorocebus aethiops , Proteínas del Citoesqueleto , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Glicoproteínas , Células HeLa , Humanos , Mamíferos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Lugares Marcados de Secuencia , Transfección , Dedos de Zinc , ZixinaRESUMEN
We summarize recent findings on the organization of the protein actin in eucaryotic cells. In particular we focus on how actin can be used to generate a vectorial force that is required for cell movement. These forces arise from protein molecules that recruit actin to the plasma membrane in such a manner that actin filaments extend outward from the cell body. This type of actin dependent force generation has been described in a nucleation-release model, which is one of several models currently being tested to explain actin dependent cell movement. Data in support of this model has arisen unexpectedly from studies of an intracellular bacteria, Listeria monocytogenes. This bacteria uses actin to propel itself during infection of eucaryotic cells. By studying Listeria movement, the roles of several eucaryotic actin interacting proteins have been identified. One of these is zyxin, a human protein that shares important structural and possibly functional properties with ActA, an actin dependent force generating protein of Listeria. We intend to test the function of these and other actin interacting proteins in a simplified system that should facilitate precise measurement of their properties of force generation in vitro.
Asunto(s)
Actinas/fisiología , Movimiento Celular/fisiología , Listeria/fisiología , Proteínas de Microfilamentos/fisiología , Actinas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/fisiología , Membrana Celular/metabolismo , Membrana Celular/fisiología , Proteínas del Citoesqueleto , Glicoproteínas , Humanos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Metaloproteínas/metabolismo , Metaloproteínas/fisiología , ZixinaRESUMEN
Here we discuss members of a new family of serine/threonine protein kinases with a likely role in cell cycle control. These kinases are referred to as polo-like kinases, after the prototypic founding member of the family, the polo gene product of Drosophila melanogaster. The polo kinase was originally identified in mutants that display abnormal mitotic spindle organization. Subsequently, potential homologues of Drosophila polo have been identified in yeasts (Cdc5p in Saccharomyces cerevisiae; plo1+ in Schizosaccharmoyces pombe) and in mammals (polo-like kinase 1; Plk1). Genetic and biochemical studies suggest that polo, Cdc5p and plo1+ may be required for mitotic spindle organization and, possibly, for cytokinesis. Likewise, the patterns of expression, activity and subcellular localization of Plk1 strongly suggest that this mammalian kinase functions also during mitosis, possibly in spindle assembly and function. In addition to Plk1, however, more distantly related members of the polo-like kinase family have been identified in mammalian cells, and the available data are consistent with the idea that some of these may act earlier in the cell cycle, possibly during G1. If this hypothesis is correct, different members of the polo-like kinase family would act at several points during the cell cycle, reminiscent of the behaviour of Cdk/cyclin complexes.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas de Drosophila , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Animales , Ciclo Celular/fisiología , Drosophila melanogaster/citología , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Humanos , Mitosis/fisiología , Datos de Secuencia Molecular , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Proto-Oncogénicas , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Schizosaccharomyces/citología , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Homología de Secuencia de Aminoácido , Quinasa Tipo Polo 1RESUMEN
Correct assembly and function of the mitotic spindle during cell division is essential for the accurate partitioning of the duplicated genome to daughter cells. Protein phosphorylation has long been implicated in controlling spindle function and chromosome segregation, and genetic studies have identified several protein kinases and phosphatases that are likely to regulate these processes. In particular, mutations in the serine/threonine-specific Drosophila kinase polo, and the structurally related kinase Cdc5p of Saccharomyces cerevisae, result in abnormal mitotic and meiotic divisions. Here, we describe a detailed analysis of the cell cycle-dependent activity and subcellular localization of Plk1, a recently identified human protein kinase with extensive sequence similarity to both Drosophila polo and S. cerevisiae Cdc5p. With the aid of recombinant baculoviruses, we have established a reliable in vitro assay for Plk1 kinase activity. We show that the activity of human Plk1 is cell cycle regulated, Plk1 activity being low during interphase but high during mitosis. We further show, by immunofluorescent confocal laser scanning microscopy, that human Plk1 binds to components of the mitotic spindle at all stages of mitosis, but undergoes a striking redistribution as cells progress from metaphase to anaphase. Specifically, Plk1 associates with spindle poles up to metaphase, but relocalizes to the equatorial plane, where spindle microtubules overlap (the midzone), as cells go through anaphase. These results indicate that the association of Plk1 with the spindle is highly dynamic and that Plk1 may function at multiple stages of mitotic progression. Taken together, our data strengthen the notion that human Plk1 may represent a functional homolog of polo and Cdc5p, and they suggest that this kinase plays an important role in the dynamic function of the mitotic spindle during chromosome segregation.
Asunto(s)
Ciclo Celular/fisiología , Proteínas de Drosophila , Proteínas Quinasas/metabolismo , Huso Acromático/fisiología , Adenosina Trifosfato/metabolismo , Animales , Baculoviridae , Secuencia de Bases , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Cartilla de ADN , Drosophila/enzimología , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Cinética , Mitosis , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis Sitio-Dirigida , Proteínas Quinasas/análisis , Proteínas Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Proteínas de Unión al ARN , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae , Huso Acromático/enzimología , Huso Acromático/ultraestructura , Spodoptera , Transfección , Quinasa Tipo Polo 1RESUMEN
polo and CDC5 are two genes required for passage through mitosis in Drosophila melanogaster and Saccharomyces cerevisiae, respectively. Both genes encode structurally related protein kinases that have been implicated in regulating the function of the mitotic spindle. Here, we report the characterization of a human protein kinase that displays extensive sequence similarity to Drosophila polo and S. cerevisiae Cdc5; we refer to this kinase as Plk1 (for polo-like kinase 1). The largest open reading frame of the Plk1 cDNA encodes a protein of 68,254 daltons, and a protein of this size is detected by immunoblotting of HeLa cell extracts with monoclonal antibodies raised against the C-terminal part of Plk1 expressed in Escherichia coli. Northern blot analysis of RNA isolated from human cells and mouse tissues shows that a single Plk1 mRNA of 2.3 kb is highly expressed in tissues with a high mitotic index, consistent with a possible function of Plk1 in cell proliferation. The Plk1 gene maps to position p12 on chromosome 16, a locus for which no associations with neoplastic malignancies are known. The Plk1 protein levels and its distribution change during the cell cycle, in a manner consistent with a role of Plk1 in mitosis. Thus, like Drosophila polo and S. cerevisiae Cdc5, human Plk1 is likely to function in cell cycle progression.
Asunto(s)
Proteínas de Ciclo Celular , Cromosomas Humanos Par 16 , Proteínas de Unión al ADN/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Proteínas Fúngicas/genética , Mitosis , Proteínas Quinasas/aislamiento & purificación , Proteínas Serina-Treonina Quinasas/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Carcinoma/genética , Ciclo Celular , Mapeo Cromosómico , Clonación Molecular , Regulación Neoplásica de la Expresión Génica , Biblioteca de Genes , Genes , Células HeLa , Humanos , Datos de Secuencia Molecular , Neoplasias Nasofaríngeas/genética , Sistemas de Lectura Abierta , Proteínas Quinasas/genética , Proteínas Quinasas/inmunología , Proteínas Quinasas/fisiología , Proteínas Proto-Oncogénicas , Proteínas de Unión al ARN , Proteínas de Saccharomyces cerevisiae , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Fracciones Subcelulares , Quinasa Tipo Polo 1RESUMEN
The 100,000 g supernatant from the unfertilized egg of the sea urchin Strongylocentrotus purpuratus has been fractionated on DEAE-cellulose and analysed for Ca2+-binding activity by the Chelex-100 competitive Ca2+-binding activity assay. The major peak of Ca2+-binding activity was subjected to further purification and the Ca2+-binding protein responsible for this Ca2+-binding-activity peak has been isolated and characterized. Non-denaturing polyacrylamide-gel electrophoresis (PAGE) followed by 45Ca2+ autoradiography suggested a molecular mass of 80 kDa for the Ca2+-binding protein. SDS/PAGE revealed that the 80 kDa protein consisted of a 1:1 molar complex of proteins of 50 and 42 kDa. The 42 kDa protein was identified as actin. The complex was not dissociated by extensive dialysis against an EGTA-containing buffer. The EGTA-stable complex was named '50K-A'.
Asunto(s)
Actinas/metabolismo , Proteínas del Huevo/metabolismo , Actinas/aislamiento & purificación , Animales , Calcio/metabolismo , Proteínas del Huevo/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Fertilización , Sustancias Macromoleculares , Óvulo/metabolismo , Erizos de MarRESUMEN
In the preceding paper [Golsteyn & Waisman (1989) Biochem. J. 257, 809-815] an EGTA-stable, Ca2+-binding heterodimer comprised of a 50 kDa protein and actin called '50K-A' was identified in the unfertilized eggs of the sea urchin Strongylocentrotus purpuratus. In the present paper we have documented the binding of 50K-A to DNAase I and the effect of 50K-A on the kinetics of actin polymerization. When 50K-A was added to pyrene-labelled rabbit skeletal-muscle actin and the salt concentration increased, the initial rate of actin polymerization was inhibited by a very low molar ratio of 50K-A to actin. Furthermore, the steady-state level of G-actin was increased in the presence of 50K-A, suggesting that 50K-A caps the preferred end of actin polymer, shifting the steady-state concentration to that of the non-preferred end. Dilution of F-actin to below its critical concentration into 50K-A resulted in a much slower rate of depolymerization, consistent with capping of the preferred end. In contrast with the Ca2+-dependent binding to DNAase, the effect of 50K-A on the kinetics of actin assembly and disassembly was Ca2+-independent. These results suggest that 50K-A is a novel actin-binding protein with some similarities to the severin/fragmin/gelsolin family of F-actin-capping proteins.