RESUMEN

BACKGROUND: The adenomatous polyposis coli (APC) protein is an important tumour suppressor in the colon. It promotes the destabilisation of free cytoplasmic beta-catenin (the vertebrate homologue of the Drosophila protein Armadillo), a critical effector of the Wnt signalling pathway. The beta-catenin protein is also a component of adherens junctions, linking these to the actin cytoskeleton. In Drosophila epithelial cells, the ubiquitous form of APC, known as E-APC, is associated with adherens junctions. This association appears to be necessary for E-APC to function in destabilising Armadillo. RESULTS: Using actin-depolymerising drugs, we established that an intact actin cytoskeleton is required for the association of E-APC with adherens junctions in the Drosophila embryo. From an analysis of profilin mutants, whose actin cytoskeleton is disrupted, we found that E-APC also requires actin filaments to associate with adhesive cell membranes in the ovary. Notably, conditions that delocalised E-APC from membranes, including a mutation in E-APC itself, caused partial detachment of Armadillo from adhesive membranes. CONCLUSIONS: Actin filaments are continuously required for E-APC to be associated with junctional membranes. These filaments may serve as tracks for E-APC to reach the adherens junctions. The failure of E-APC to do so appears to affect the integrity of junctional complexes.

Asunto(s)

Citoesqueleto de Actina/fisiología , Actinas/metabolismo , Uniones Adherentes/metabolismo , Membrana Celular/metabolismo , Proteínas Contráctiles , Proteínas del Citoesqueleto/metabolismo , Proteínas de Drosophila , Proteínas de Insectos/metabolismo , Transactivadores , Citoesqueleto de Actina/efectos de los fármacos , Proteína de la Poliposis Adenomatosa del Colon , Animales , Proteínas del Dominio Armadillo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Tamaño de la Célula , Colchicina/farmacología , Citocalasina D/farmacología , Drosophila melanogaster/embriología , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Femenino , Colorantes Fluorescentes/metabolismo , Proteínas de Insectos/inmunología , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Microscopía Confocal , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Ovario/citología , Ovario/metabolismo , Faloidina/farmacología , Profilinas , Tiazoles/farmacología , Tiazolidinas , Factores de Transcripción

RESUMEN

Cdc20/fizzy family proteins are involved in activation of the anaphase-promoting complex/cyclosome, which catalyzes the ubiquitin-dependent proteolysis of cell cycle regulatory proteins such as anaphase inhibitors and mitotic cyclins, leading to chromosome segregation and exit from mitosis. Previous work has shown that human Cdc20 (hCdc20/p55CDC) associates with one or more kinases. We report here that Cdc20-associated myelin basic protein kinase activity peaks sharply in early M phase (embryonic cells) or in G2 phase (somatic cells). In HeLa cells, Cdc20 is associated with the kinase aurora2/Aik. Aurora2/Aik is a member of the aurora/Ipl1 family of kinases that, like Cdc20, previously has been shown to be localized at mitotic spindle poles and is involved in regulating chromosome segregation and maintaining genomic stability. The demonstration that Cdc20 is associated with aurora2/Aik suggests that some function of Cdc20 is carried out or regulated through its association with aurora2/Aik.

Asunto(s)

Proteínas de Ciclo Celular/fisiología , Ciclo Celular/fisiología , Proteínas Quinasas/fisiología , Proteínas Serina-Treonina Quinasas , Proteínas de Saccharomyces cerevisiae , Aurora Quinasas , Proteínas Cdc20 , Femenino , Fertilización , Células HeLa , Humanos , Datos de Secuencia Molecular , Oocitos , Transducción de Señal

RESUMEN

Progression through the cell cycle is regulated in part by the sequential activation and inactivation of cyclin-dependent kinases (CDKs). Many signals arrest the cell cycle through inhibition of CDKs by CDK inhibitors (CKIs). p27(Kip1) (p27) was first identified as a CKI that binds and inhibits cyclin A/CDK2 and cyclin E/CDK2 complexes in G1. Here we report that p27 has an additional property, the ability to induce a proteolytic activity that cleaves cyclin A, yielding a truncated cyclin A lacking the mitotic destruction box. Other CKIs (p15(Ink4b), p16(Ink4a), p21(Cip1), and p57(Kip2)) do not induce cleavage of cyclin A; other cyclins (cyclin B, D1, and E) are not cleaved by the p27-induced protease activity. The C-terminal half of p27, which is dispensable for its kinase inhibitory activity, is required to induce cleavage. Mechanistically, p27 does not appear to cause cleavage through direct interaction with cyclin/CDK complexes. Instead, it activates a latent protease that, once activated, does not require the continuing presence of p27. Mutation of cyclin A at R70 or R71, residues at or very close to the cleavage site, blocks cleavage. Noncleavable mutants are still recognized by the anaphase-promoting complex/cyclosome pathway responsible for ubiquitin-dependent proteolysis of mitotic cyclins, indicating that the p27-induced cleavage of cyclin A is part of a separate pathway. We refer to this protease as Tsap (pTwenty-seven- activated protease).

Asunto(s)

Proteínas de Ciclo Celular , Ciclina A/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Supresoras de Tumor , Células 3T3 , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Ciclina A/química , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , ADN Complementario , Eliminación de Gen , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Plásmidos , Proteínas Recombinantes/metabolismo , Reticulocitos/metabolismo , Transducción de Señal

RESUMEN

A key player in mitotic progression is a cell-cycle-regulated ubiquitin-protein ligase complex known as the anaphase-promoting complex or cyclosome (APC/C). The APC/C is part of the machinery that promotes the metaphase-anaphase transition by mediating the ubiquitin-dependent destruction of anaphase inhibitors and initiates exit from mitosis by degrading mitotic cyclins. This review describes the known components and substrates of the mitotic ubiquitination machinery and discusses how a new subfamily of proteins that contain the WD40 repeat (the Fizzy/Cdc20p family) might activate the APC/C to allow temporal differences in substrate ubiquitination during progression through mitosis.

Asunto(s)

Ligasas/fisiología , Mitosis/fisiología , Complejos de Ubiquitina-Proteína Ligasa , Ubiquitinas/fisiología , Levaduras/enzimología , Ciclosoma-Complejo Promotor de la Anafase , Proteínas Fúngicas/fisiología , Ubiquitina-Proteína Ligasas , Levaduras/citología

RESUMEN

UBC11 is the Saccharomyces cerevisiae gene that is most similar in sequence to E2-C, a ubiquitin carrier protein required for the destruction of mitotic cyclins and proteins that maintain sister chromatid cohesion in animal cells and in Schizosaccharomyces pombe. We have disrupted the UBC11 gene and found it is not essential for yeast cell viability even when combined with deletion of UBC4, a gene that has also been implicated in mitotic cyclin destruction. Ubc11p does not ubiquitinate cyclin B in clam cell-free extracts in vitro and the destruction of Clb2p is not impaired in extracts prepared from delta ubc11 or delta ubc4 delta ubc11 cells. These results suggest Ubc4p and Ubc11p together are not essential for mitotic cyclin destruction in S. cerevisiae and we can find no evidence to suggest that Ubc11p is the true functional homologue of E2-C.

Asunto(s)

Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Enzimas Ubiquitina-Conjugadoras , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Bivalvos/enzimología , Western Blotting , Ciclo Celular/genética , Ciclina B/metabolismo , Cisteína , Eliminación de Gen , Genotipo , Humanos , Ligasas/genética , Ligasas/metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Ubiquitinas/metabolismo

RESUMEN

Destruction of mitotic cyclins by ubiquitin-dependent proteolysis is required for cells to complete mitosis and enter interphase of the next cell cycle. In clam eggs, this process is catalyzed by a cyclin-selective ubiquitin carrier protein, E2-C, and the cyclosome/anaphase promoting complex (APC), a 20S particle containing cyclin-selective ubiquitin ligase activity. Here we report cloning a human homolog of E2-C, UbcH10, which shares 61% amino acid identity with clam E2-C and can substitute for clam E2-C in vitro. Dominant-negative clam E2-C and human UbcH10 proteins, created by altering the catalytic cysteine to serine, inhibit the in vitro ubiquitination and destruction of cyclin B in clam oocyte extracts. When transfected into mammalian cells, mutant UbcH10 inhibits the destruction of both cyclin A and B, arrests cells in M phase, and inhibits the onset of anaphase, presumably by blocking the ubiquitin-dependent proteolysis of proteins responsible for sister chromatid separation. Thus, E2-C/UbcH10-mediated ubiquitination is involved in both cdc2 inactivation and sister chromatid separation, processes that are normally coordinated during exit from mitosis.

Asunto(s)

Proteínas Portadoras/metabolismo , Ciclo Celular , Ligasas/metabolismo , Enzimas Ubiquitina-Conjugadoras , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bivalvos , Proteínas Portadoras/química , Ciclinas/metabolismo , Cartilla de ADN , Femenino , Células HeLa , Humanos , Cinética , Ligasas/biosíntesis , Ligasas/química , Metafase , Mitosis , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oocitos/fisiología , Mutación Puntual , Reacción en Cadena de la Polimerasa , Ranidae , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Homología de Secuencia de Aminoácido

RESUMEN

The ERD2 gene of Saccharomyces cerevisiae encodes the receptor which retrieves HDEL-containing containing ER proteins from the Golgi apparatus. Viable erd2 mutants have been isolated that show no obvious HDEL-dependent retention of the luminal ER protein BiP, suggesting that retrieval of HDEL proteins is not essential for growth. However, cells that lack Erd2p completely have a defective Golgi apparatus and cannot grow. This observation led to the suggestion that the receptor had a second function, possibly related to its ability to recycle from Golgi to ER. In this paper we investigate the requirements for Erd2p to support growth. We show that mutations that block its recycling also prevent growth. In addition, we show that all mutant receptors that can support growth have a residual ability to retrieve BiP, which is detectable when they are overexpressed. Mere recycling of an inactive form of the receptor, mediated by a cytoplasmic KKXX sequence, is not sufficient for growth. Furthermore, saturation of the receptor by expression of an HDEL-tagged version of pro-alpha factor inhibits growth, even of strains that do not show obvious BiP retention. We conclude that growth requires the HDEL-dependent retrieval of one or more proteins, and that these proteins can be recognized even under conditions where BiP is secreted. Genetic screens have failed to identify any one protein whose loss could account for the Erd2p requirement. Therefore, a growth may require the retention of multiple HDEL proteins in the ER, or alternatively the removal of such proteins from the Golgi apparatus.

Asunto(s)

Proteínas Fúngicas/metabolismo , Proteínas HSP70 de Choque Térmico , Proteínas de la Membrana/metabolismo , Oligopéptidos/fisiología , Receptores de Péptidos/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/crecimiento & desarrollo , Secuencia de Aminoácidos , Genes Fúngicos/genética , Aparato de Golgi/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de la Membrana/genética , Modelos Biológicos , Datos de Secuencia Molecular , Mutación/fisiología , Oligopéptidos/genética , Señales de Clasificación de Proteína/genética , Receptores de Péptidos/fisiología , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/metabolismo

Asunto(s)

Retículo Endoplásmico/metabolismo , Proteínas Fúngicas/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Señales de Clasificación de Proteína/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Transporte Biológico , Compartimento Celular , Citoplasma/metabolismo , Datos de Secuencia Molecular , Receptores de Péptidos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/ultraestructura

RESUMEN

The KDEL receptor is a seven-transmembrane-domain protein that is responsible for the retrieval of endoplasmic reticulum (ER) proteins from the Golgi complex. It is a temporary resident of the Golgi apparatus: upon binding a KDEL-containing ligand, it moves to the ER, where the ligand is released. We have expressed mutant forms of the human receptor in COS cells and examined their intracellular locations and ligand-binding capacities. We show that ligand binding is dependent on charged residues within the transmembrane domains. Surprisingly, retrograde transport of occupied receptor is unaffected by most mutations in the cytoplasmic loops, but is critically dependent upon an aspartic acid residue in the seventh transmembrane domain. Retention in the Golgi apparatus requires neither ligand binding nor this aspartate residue, and thus is independent of receptor recycling. We suggest that movement of the receptor is controlled by conformational changes and intermolecular interactions within the membrane bilayer.

Asunto(s)

Aparato de Golgi/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores de Péptidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Transporte Biológico , Células Cultivadas , Citoplasma/metabolismo , Análisis Mutacional de ADN , Retículo Endoplásmico/metabolismo , Humanos , Ligandos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética