RESUMEN
The endocytic protein NUMB has been implicated in the control of various polarized cellular processes, including the acquisition of mesenchymal migratory traits through molecular mechanisms that have only been partially defined. Here, we report that NUMB is a negative regulator of a specialized set of understudied, apically restricted, actin-based protrusions, the circular dorsal ruffles (CDRs), induced by either PDGF or HGF stimulation. Through its PTB domain, NUMB binds directly to an N-terminal NPLF motif of the ARF6 guanine nucleotide exchange factor, EFA6B, and promotes its exchange activity in vitro. In cells, a NUMB-EFA6B-ARF6 axis regulates the recycling of the actin regulatory cargo RAC1 and is critical for the formation of CDRs that mark the acquisition of a mesenchymal mode of motility. Consistently, loss of NUMB promotes HGF-induced cell migration and invasion. Thus, NUMB negatively controls membrane protrusions and the acquisition of mesenchymal migratory traits by modulating EFA6B-ARF6 activity.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Movimiento Celular/fisiología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de la Membrana/metabolismo , Mesodermo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Factor 6 de Ribosilación del ADP , Línea Celular Tumoral , Polaridad Celular , Células HeLa , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Proteínas de la Membrana/genética , Mesodermo/citología , Proteínas del Tejido Nervioso/genética , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Unión Proteica , Dominios Proteicos , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The budding yeast Saccharomyces cerevisiae is a very powerful genetic model that has been extensively used in cell cycle studies. Despite the fact that its small size has made imaging studies challenging (haploid cells have a diameter of approximately 4-5 µm that is very close to the maximal optical microscope resolution, ca. 0.20-0.25 µm), the continual improvement of imaging tags and techniques has made it possible to visualize organelles and macromolecules also in this organism. The possibility to easily epitope-tag endogenous proteins and follow them during synchronized cell cycles has proved critical for understanding the distribution of Mitotic Exit Network (MEN) components and gathering insights into their regulation. In this chapter, we describe a detailed protocol for indirect immunofluorescence of fixed cells outlining fixation strategies, cell wall digestion, and the use of primary and secondary antibodies conjugated to fluorescent moieties. This protocol can be used to successfully localize endogenously expressed yeast proteins including MEN components.
Asunto(s)
Técnica del Anticuerpo Fluorescente Indirecta/métodos , Microscopía Fluorescente/métodos , Saccharomyces cerevisiae/citología , Ciclo Celular , Proteínas de Ciclo Celular/análisis , Mitosis , Proteínas Serina-Treonina Quinasas/análisis , Proteínas Tirosina Fosfatasas/análisis , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/análisis , Tubulina (Proteína)/análisisRESUMEN
Protein interaction modules coordinate the connections within and the activity of intracellular signaling networks. The Eps15 Homology (EH) module, a protein-protein interaction domain that is a key feature of the EH-network, was originally identified in a few proteins involved in endocytosis and vesicle trafficking, and has subsequently also been implicated in actin reorganization, nuclear shuttling, and DNA repair. Here we report an extensive characterization of the physical connections and of the functional wirings of the EH-network in the nematode. Our data show that one of the major physiological roles of the EH-network is in neurotransmission. In addition, we found that the proteins of the network intersect, and possibly coordinate, a number of "territories" of cellular activity including endocytosis/recycling/vesicle transport, actin dynamics, general metabolism and signal transduction, ubiquitination/degradation of proteins, DNA replication/repair, and miRNA biogenesis and processing.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Regulación de la Expresión Génica , Estructura Terciaria de Proteína , Reproducibilidad de los Resultados , Transmisión Sináptica , Técnicas del Sistema de Dos HíbridosRESUMEN
NUMB is a cell fate determinant, which, by asymmetrically partitioning at mitosis, controls cell fate choices by antagonising the activity of the plasma membrane receptor of the NOTCH family. NUMB is also an endocytic protein, and the NOTCH-NUMB counteraction has been linked to this function. There might be, however, additional functions of NUMB, as witnessed by its proposed role as a tumour suppressor in breast cancer. Here we describe a previously unknown function for human NUMB as a regulator of tumour protein p53 (also known as TP53). NUMB enters in a tricomplex with p53 and the E3 ubiquitin ligase HDM2 (also known as MDM2), thereby preventing ubiquitination and degradation of p53. This results in increased p53 protein levels and activity, and in regulation of p53-dependent phenotypes. In breast cancers there is frequent loss of NUMB expression. We show that, in primary breast tumour cells, this event causes decreased p53 levels and increased chemoresistance. In breast cancers, loss of NUMB expression causes increased activity of the receptor NOTCH. Thus, in these cancers, a single event-loss of NUMB expression-determines activation of an oncogene (NOTCH) and attenuation of the p53 tumour suppressor pathway. Biologically, this results in an aggressive tumour phenotype, as witnessed by findings that NUMB-defective breast tumours display poor prognosis. Our results uncover a previously unknown tumour suppressor circuitry.
Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Células Cultivadas , Daño del ADN , Resistencia a Antineoplásicos , Silenciador del Gen , Humanos , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Pronóstico , Unión Proteica , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , UbiquitinaciónRESUMEN
Spinocerebellar ataxia type 3 is a human neurodegenerative disease resulting from polyglutamine tract expansion. The affected protein, ataxin-3, which contains an N-terminal Josephin domain followed by tandem ubiquitin (Ub)-interacting motifs (UIMs) and a polyglutamine stretch, has been implicated in the function of the Ub proteasome system. NMR-based structural analysis has now revealed that the Josephin domain binds Ub and has a papain-like fold that is reminiscent of that of other deubiquitinases, despite primary sequence divergence but consistent with its deubiqutinating activity. Mutation of the catalytic Cys enhances the stability of a complex between ataxin-3 and polyubiquitinated proteins. This effect depends on the integrity of the UIM region, suggesting that the UIMs are bound to the substrate polyubiquitin during catalysis. We propose that ataxin-3 functions as a polyubiquitin chain-editing enzyme.
Asunto(s)
Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Ubiquitina/metabolismo , Ataxina-3 , Catálisis , Humanos , Modelos Moleculares , Proteínas del Tejido Nervioso/genética , Resonancia Magnética Nuclear Biomolecular , Proteínas Nucleares , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas RepresorasRESUMEN
The small GTP-binding protein Rab11 is an essential regulator of the dynamics of recycling endosomes. Here we report the crystallographic analysis of the GDP/GTP cycle of human Rab11a, and a structure-based mutagenesis study that identifies a novel mutant phenotype. The crystal structures show that the nucleotide-sensitive switch 1 and 2 regions differ from those of other Rab proteins. In Rab11-GDP, they contribute to a close packed symmetrical dimer, which may associate to membranes in the cell and allow Rab11 to undergo GDP/GTP cycles without recycling to the cytosol. The structure of active Rab11 delineates a three-dimensional site that includes switch 1 and is separate from the site defined by the Rab3/Rabphilin interface. It is proposed to form a novel interface for a Rab11 partner compatible with the simultaneous binding of another partner at the Rabphilin interface. Mutation of Ser(29) to Phe in this epitope resulted in morphological modifications of the recycling compartment that are distinct from those induced by the classical dominant-negative and constitutively active Rab11 mutants. Recycling endosomes condensed in the perinuclear region where they retained recycling transferrin, and they clustered Rab11- and EEA1-positive membranes. Altogether, our study suggests that this mutation impairs a specific subset of Rab11 interactions, possibly those involved in cytoskeleton-based movements driving the slow recycling pathway.
Asunto(s)
Endosomas/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Transporte Biológico , Clonación Molecular , Cartilla de ADN , Guanosina Difosfato/química , Guanosina Trifosfato/química , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Transferrina/metabolismo , Proteínas de Unión al GTP rab/genéticaRESUMEN
Rho GTPases are key regulators of actin dynamics. We report that the Rho GTPase TCL, which is closely related to Cdc42 and TC10, localizes to the plasma membrane and the early/sorting endosomes in HeLa cells, suggesting a role in the early endocytic pathway. Receptor-dependent internalization of transferrin (Tf) is unaffected by suppression of endogenous TCL by small interfering RNA treatment. However, Tf accumulates in Rab5-positive uncoated endocytic vesicles and fails to reach the early endosome antigen-1-positive early endosomal compartments and the pericentriolar recycling endosomes. Moreover, Tf release upon TCL knockdown is significantly slower. Conversely, in the presence of dominant active TCL, internalized Tf accumulates in early endosome antigen-1-positive early/sorting endosomes and not in perinuclear recycling endosomes. Tf recycles directly from the early/sorting endosomes and it is normally released by the cells. The same phenotype is generated by replacing the C terminus of dominant active Cdc42 and TC10 with that of TCL, indicating that all three proteins share downstream effector proteins. Thus, TCL is essential for clathrin-dependent endocytosed receptors to enter the early/sorting endosomes. Furthermore, the active GTPase favors direct recycling from early/sorting endosomes without accumulating in the perinuclear recycling endosomes.
Asunto(s)
Membrana Celular/enzimología , Endocitosis/fisiología , Endosomas/enzimología , GTP Fosfohidrolasas/metabolismo , GTP Fosfohidrolasas/efectos de los fármacos , Células HeLa , Humanos , Inmunohistoquímica , Unión Proteica , Estructura Terciaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/fisiología , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fracciones Subcelulares , Transferrina/metabolismo , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rab5 , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
The Sec7 domain guanine nucleotide exchange factors (GEFs) for the GTPase ARF are highly conserved regulators of membrane dynamics and protein trafficking. The interactions of large ARF GEFs with cellular membranes for localization and/or activation are likely to participate in regulated recruitment of ARF and effectors. However, these interactions remain largely unknown. Here we characterize Gmh1p, the first Golgi transmembrane-domain partner of any of the high-molecular-weight ARF-GEFs. Gmh1p is an evolutionarily conserved protein. We demonstrate molecular interaction between the yeast Gmh1p and the large ARF-GEFs Gea1p and Gea2p. This interaction involves a domain of Gea1p and Gea2p that is conserved in the eukaryotic orthologues of the Gea proteins. A single mutation in a conserved amino acid residue of this domain is sufficient to abrogate the interaction, whereas the overexpression of Gmh1p can compensate in vivo defects caused by mutations in this domain. We show that Gmh1p is an integral membrane protein that localizes to the early Golgi in yeast and in human HeLa cells and cycles through the ER. Hence, we propose that Gmh1p acts as a positive Golgi-membrane partner for Gea function. These results are of general interest given the evolutionary conservation of both ARF-GEFs and the Gmh proteins.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Aparato de Golgi/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factor 1 de Ribosilacion-ADP/genética , Factor 1 de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Retículo Endoplásmico/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Células HeLa , Humanos , Mutación , Estructura Terciaria de Proteína , Levaduras/genética , Levaduras/metabolismoRESUMEN
Rab4 and Rab11 are small GTPases belonging to the Ras superfamily. They both function as regulators along the receptor recycling pathway. We have identified a novel 80-kDa protein that interacts specifically with the GTP-bound conformation of Rab4, and subsequent work has shown that it also interacts strongly with Rab11. We name this protein Rab coupling protein (RCP). RCP is predominantly membrane-bound and is expressed in all cell lines and tissues tested. It colocalizes with early endosomal markers including Rab4 and Rab11 as well as with the transferrin receptor. Overexpression of the carboxyl-terminal region of RCP, which contains the Rab4- and Rab11-interacting domain, results in a dramatic tubulation of the transferrin compartment. Furthermore, expression of this mutant causes a significant reduction in endosomal recycling without affecting ligand uptake or degradation in quantitative assays. RCP is a homologue of Rip11 and therefore belongs to the recently described Rab11-FIP family.