RESUMEN

Disruption of epithelial organization and loss of growth control are universal features of carcinomas, yet how these features are linked during cancer progression remains poorly understood. Cell polarity proteins control cellular and tissue organization and are emerging as important mediators of cancer progression. The Par3 polarity protein is a molecular scaffold that functions to recruit and spatially organize signaling factors, and was recently identified as a suppressor of breast cancer invasion and metastasis. Here, we show that loss of Par3 in mammary epithelial cells promotes apoptosis, and that oncogenic Notch overcomes the apoptotic signal to reveal an unexpected pro-proliferative role for loss of Par3 in mammary tumors. In this context, loss of Par3 deregulates Rac1 activity to activate Jun N-terminal Kinase-dependent proliferation and tumor growth. Thus, we demonstrate a mechanism by which loss of Par3 promotes proliferation and tumorigenesis, which supports a tumor-suppressive function for Par3 in the mammary epithelium.

Asunto(s)

Apoptosis , Moléculas de Adhesión Celular/genética , Transformación Celular Neoplásica/genética , Sistema de Señalización de MAP Quinasas , Neuropéptidos , Proteína de Unión al GTP rac1 , Proteínas Adaptadoras Transductoras de Señales , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Moléculas de Adhesión Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular , Polaridad Celular/efectos de los fármacos , Polaridad Celular/genética , Transformación Celular Neoplásica/efectos de los fármacos , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Femenino , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/patología , Ratones , Neuropéptidos/antagonistas & inhibidores , Neuropéptidos/genética , Neuropéptidos/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patología , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo

RESUMEN

A defining characteristic of eukaryotic cells is the possession of a nuclear envelope. Transport of macromolecules between the nuclear and cytoplasmic compartments occurs through nuclear pore complexes that span the double membrane of this envelope. The molecular basis for transport has been revealed only within the last few years. The transport mechanism lacks motors and pumps and instead operates by a process of facilitated diffusion of soluble carrier proteins, in which vectoriality is provided by compartment-specific assembly and disassembly of cargo-carrier complexes. The carriers recognize localization signals on the cargo and can bind to pore proteins. They also bind a small GTPase, Ran, whose GTP-bound form is predominantly nuclear. Ran-GTP dissociates import carriers from their cargo and promotes the assembly of export carriers with cargo. The ongoing discovery of numerous carriers, Ran-independent transport mechanisms, and cofactors highlights the complexity of the nuclear transport process. Multiple regulatory mechanisms are also being identified that control cargo-carrier interactions. Circadian rhythms, cell cycle, transcription, RNA processing, and signal transduction are all regulated at the level of nucleocytoplasmic transport. This review focuses on recent discoveries in the field, with an emphasis on the carriers and cofactors involved in transport and on possible mechanisms for movement through the nuclear pores.

Asunto(s)

Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Poro Nuclear/metabolismo , Secuencia de Aminoácidos , Transporte Biológico/fisiología , Datos de Secuencia Molecular , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteína de Unión al GTP ran/fisiología

RESUMEN

The C. elegans genes ced-2, ced-5, and ced-10, and their mammalian homologs crkII, dock180, and rac1, mediate cytoskeletal rearrangements during phagocytosis of apoptotic cells and cell motility. Here, we describe an additional member of this signaling pathway, ced-12, and its mammalian homologs, elmo1 and elmo2. In C. elegans, CED-12 is required for engulfment of dying cells and for cell migrations. In mammalian cells, ELMO1 functionally cooperates with CrkII and Dock180 to promote phagocytosis and cell shape changes. CED-12/ELMO-1 binds directly to CED-5/Dock180; this evolutionarily conserved complex stimulates a Rac-GEF, leading to Rac1 activation and cytoskeletal rearrangements. These studies identify CED-12/ELMO as an upstream regulator of Rac1 that affects engulfment and cell migration from C. elegans to mammals.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/fisiología , Proteínas Portadoras/metabolismo , Movimiento Celular/fisiología , Proteínas del Citoesqueleto , Proteínas del Helminto/metabolismo , Fagocitosis/fisiología , Proteínas Proto-Oncogénicas , Proteínas de Unión al GTP rac/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Proteínas Reguladoras de la Apoptosis , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Línea Celular , Extensiones de la Superficie Celular/metabolismo , Citoesqueleto/metabolismo , Citometría de Flujo , Genes de Helminto , Genes Reporteros , Gónadas/crecimiento & desarrollo , Proteínas del Helminto/genética , Humanos , Masculino , Ratones , Microscopía Fluorescente , Datos de Secuencia Molecular , Proteínas Quinasas/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-crk , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Transducción de Señal/fisiología , Distribución Tisular

RESUMEN

The Cdc42 GTPase binds to numerous effector proteins that control cell polarity, cytoskeletal remodelling and vesicle transport. In many cases the signalling pathways downstream of these effectors are not known. Here we show that the Cdc42 effectors Borg1 to Borg3 bind to septin GTPases. Endogenous septin Cdc10 and Borg3 proteins can be immunoprecipitated together by an anti-Borg3 antibody. The ectopic expression of Borgs disrupts normal septin organization. Cdc42 negatively regulates this effect and inhibits the binding of Borg3 to septins. Borgs are therefore the first known regulators of mammalian septin organization and provide an unexpected link between the septin and Cdc42 GTPases.

Asunto(s)

Proteínas Sanguíneas/metabolismo , Activadores de GTP Fosfohidrolasa , GTP Fosfohidrolasas/metabolismo , Reguladores de Proteínas de Unión al GTP , Proteína de Unión al GTP cdc42/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Sanguíneas/química , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Proteínas del Citoesqueleto , Ratones , Microscopía Fluorescente , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Proteínas de Unión al ARN , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Unión al GTP rho

RESUMEN

Recent studies indicate that insulin stimulation of glucose transporter (GLUT)4 translocation requires at least two distinct insulin receptor-mediated signals: one leading to the activation of phosphatidylinositol 3 (PI-3) kinase and the other to the activation of the small GTP binding protein TC10. We now demonstrate that TC10 is processed through the secretory membrane trafficking system and localizes to caveolin-enriched lipid raft microdomains. Although insulin activated the wild-type TC10 protein and a TC10/H-Ras chimera that were targeted to lipid raft microdomains, it was unable to activate a TC10/K-Ras chimera that was directed to the nonlipid raft domains. Similarly, only the lipid raft-localized TC10/ H-Ras chimera inhibited GLUT4 translocation, whereas the TC10/K-Ras chimera showed no significant inhibitory activity. Furthermore, disruption of lipid raft microdomains by expression of a dominant-interfering caveolin 3 mutant (Cav3/DGV) inhibited the insulin stimulation of GLUT4 translocation and TC10 lipid raft localization and activation without affecting PI-3 kinase signaling. These data demonstrate that the insulin stimulation of GLUT4 translocation in adipocytes requires the spatial separation and distinct compartmentalization of the PI-3 kinase and TC10 signaling pathways.

Asunto(s)

Insulina/metabolismo , Microdominios de Membrana/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Proteínas de Unión al GTP rho/metabolismo , Adipocitos/citología , Secuencia de Aminoácidos , Animales , Caveolas , Caveolina 1 , Caveolinas/genética , Caveolinas/aislamiento & purificación , Células Cultivadas , Transportador de Glucosa de Tipo 4 , Ratones , Datos de Secuencia Molecular , Mutación , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Proteínas ras/genética , Proteínas de Unión al GTP rho/genética

RESUMEN

Crm1 is a member of the karyopherin family of nucleocytoplasmic transport receptors and mediates the export of proteins from the nucleus by forming a ternary complex with cargo and Ran:GTP. This complex translocates through the nuclear pores and dissociates in the cytosol. The yeast protein Yrb2p participates in this pathway and binds Crm1, but its mechanism of action has not been established. We show that the human orthologue of Yrb2p, Ran-binding protein 3 (RanBP3), acts as a cofactor for Crm1-mediated export in a permeabilized cell assay. RanBP3 binds directly to Crm1, and the complex possesses an enhanced affinity for both Ran:GTP and cargo. RanBP3 shuttles between the nucleus and the cytoplasm by a Crm1-dependent mechanism, and the Crm1--RanBP3-NES-Ran:GTP quarternary complex can associate with nucleoporins. We infer that this complex translocates through the nuclear pore to the cytoplasm where it is disassembled by RanBP1 and Ran GTPase--activating protein.

Asunto(s)

Transporte Activo de Núcleo Celular/fisiología , Proteínas Portadoras/metabolismo , Carioferinas , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático , Receptores Citoplasmáticos y Nucleares , Proteínas Portadoras/genética , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Sustancias Macromoleculares , Poro Nuclear/metabolismo , Proteínas Nucleares/genética , Unión Proteica/fisiología , Estructura Terciaria de Proteína/fisiología , Saccharomyces , Especificidad por Sustrato , Técnicas del Sistema de Dos Híbridos , Proteína de Unión al GTP ran/metabolismo , Proteína Exportina 1

RESUMEN

The Ran guanosine triphosphatase (GTPase) controls nucleocytoplasmic transport, mitotic spindle formation, and nuclear envelope assembly. These functions rely on the association of the Ran-specific exchange factor, RCC1 (regulator of chromosome condensation 1), with chromatin. We find that RCC1 binds directly to mononucleosomes and to histones H2A and H2B. RCC1 utilizes these histones to bind Xenopus sperm chromatin, and the binding of RCC1 to nucleosomes or histones stimulates the catalytic activity of RCC1. We propose that the docking of RCC1 to H2A/H2B establishes the polarity of the Ran-GTP gradient that drives nuclear envelope assembly, nuclear transport, and other nuclear events.

Asunto(s)

Proteínas de Ciclo Celular , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Intercambio de Guanina Nucleótido , Histonas/metabolismo , Proteínas Nucleares , Nucleosomas/metabolismo , Transporte Activo de Núcleo Celular , Animales , Catálisis , Núcleo Celular/metabolismo , Pollos , ADN/metabolismo , Dimerización , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Masculino , Membrana Nuclear/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Espermatozoides , Proteínas de Xenopus , Xenopus laevis , Proteína de Unión al GTP ran/metabolismo

RESUMEN

AFX belongs to a subfamily of Forkhead transcription factors that are phosphorylated by protein kinase B (PKB), also known as Akt. Phosphorylation inhibits the transcriptional activity of AFX and changes the steady-state localization of the protein from the nucleus to the cytoplasm. Our goal was threefold: to identify the cellular compartment in which PKB phosphorylates AFX, to determine whether the nuclear localization of AFX plays a role in regulating its transcriptional activity, and to elucidate the mechanism by which phosphorylation alters the localization of AFX. We show that phosphorylation of AFX by PKB occurs in the nucleus. In addition, nuclear export mediated by the export receptor, Crm1, is required for the inhibition of AFX transcriptional activity. Both phosphorylated and unphosphorylated AFX, however, bind Crm1 and can be exported from the nucleus. These results suggest that export is unregulated and that phosphorylation by PKB is not required for the nuclear export of AFX. We show that AFX enters the nucleus by an active, Ran-dependent mechanism. Amino acids 180 to 221 of AFX comprise a nonclassical nuclear localization signal (NLS). S193, contained within this atypical NLS, is a PKB-dependent phosphorylation site on AFX. Addition of a negative charge at S193 by mutating the residue to glutamate reduces nuclear accumulation. PKB-mediated phosphorylation of AFX, therefore, attenuates the import of the transcription factor, which shifts the localization of the protein from the nucleus to the cytoplasm and results in the inhibition of AFX transcriptional activity.

Asunto(s)

Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/fisiología , Factores de Transcripción/fisiología , Células 3T3 , Animales , Transporte Biológico/fisiología , Proteínas de Ciclo Celular , Núcleo Celular/fisiología , Factores de Transcripción Forkhead , Ratones , Fosforilación , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Transcripción Genética

RESUMEN

Rabphilin is a secretory vesicle protein that interacts with the GTP-bound form of the small GTPase Rab3. We investigated the involvement of Rabphilin in endocytosis using different point mutants of the protein. Overexpression of wild-type Rabphilin in the insulin-secreting cell line HIT-T15 did not affect receptor-mediated transferrin endocytosis. By contrast, Rabphilin V61A, a mutant that is unable to interact with Rab3, enhanced the rate of transferrin internalization. The effect of Rabphilin V61A was not mimicked by Rabphilin L83A, another mutant with impaired Rab3 binding. Careful analysis of the properties of the two mutants revealed that Rabphilin V61A and Rabphilin L83A are both targeted to secretory vesicles, have stimulatory activity on exocytosis, and bind equally well to alpha-actinin. However, Rabphilin L83A fails to interact with Rabaptin-5, an important component of the endocytotic machinery. These results indicate that Rabphilin promotes receptor-mediated endocytosis and that its action is negatively modulated by Rab3. We propose that the hydrolysis of GTP that is coupled to the exocytotic event disrupts the Rabphilin-Rab3 complex and permits the recruitment of Rabaptin-5 at the fusion site. Our data show that immediately after internalization the transferrin receptor and VAMP-2 colocalize on the same vesicular structures, suggesting that Rabphilin favors the rapid recycling of the components of the secretory vesicle.

Asunto(s)

Endocitosis , Proteínas de Unión al GTP/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte Vesicular , Proteínas de Unión al GTP rab3/metabolismo , Línea Celular , Membrana Celular/metabolismo , Unión Proteica

RESUMEN

The stimulation of glucose uptake by insulin in muscle and adipose tissue requires translocation of the GLUT4 glucose transporter protein from intracellular storage sites to the cell surface. Although the cellular dynamics of GLUT4 vesicle trafficking are well described, the signalling pathways that link the insulin receptor to GLUT4 translocation remain poorly understood. Activation of phosphatidylinositol-3-OH kinase (PI(3)K) is required for this trafficking event, but it is not sufficient to produce GLUT4 translocation. We previously described a pathway involving the insulin-stimulated tyrosine phosphorylation of Cbl, which is recruited to the insulin receptor by the adapter protein CAP. On phosphorylation, Cbl is translocated to lipid rafts. Blocking this step completely inhibits the stimulation of GLUT4 translocation by insulin. Here we show that phosphorylated Cbl recruits the CrkII-C3G complex to lipid rafts, where C3G specifically activates the small GTP-binding protein TC10. This process is independent of PI(3)K, but requires the translocation of Cbl, Crk and C3G to the lipid raft. The activation of TC10 is essential for insulin-stimulated glucose uptake and GLUT4 translocation. The TC10 pathway functions in parallel with PI(3)K to stimulate fully GLUT4 translocation in response to insulin.

Asunto(s)

Adipocitos/metabolismo , Proteínas del Citoesqueleto/metabolismo , Glucosa/metabolismo , Insulina/farmacología , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Ubiquitina-Proteína Ligasas , Proteínas de Unión al GTP rho/metabolismo , Animales , Células CHO , Línea Celular , Membrana Celular/metabolismo , Cricetinae , Activación Enzimática , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 4 , Factor 2 Liberador de Guanina Nucleótido/metabolismo , Microdominios de Membrana/metabolismo , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Quinasas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-cbl , Proteínas Proto-Oncogénicas c-crk , Proteínas Recombinantes de Fusión/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rho/genética

RESUMEN

TC10 is a member of the Rho family of GTPases, most closely related to Cdc42. This family of proteins mediates cytoskeletal rearrangements, activation of signal transduction cascades, and activation of gene transcription. A current focus is to identify and characterize the GTPase effectors that are involved in these cellular events. Many specific effectors for Cdc42 have been identified, most of which bind equally well to TC10, though a subset has only a low affinity for TC10. No protein that specifically interacts with TC10 has yet been described. Here, we report the cloning and characterization of PIST, a TC10-specific interacting protein. PIST possesses a PDZ domain and two, putative, coiled-coil domains, one of which contains a leucine zipper. It interacts directly and specifically with TC10:GTP, though with low affinity, and a mutation within the effector binding domain of TC10 disrupts the interaction. PIST also forms homodimers. The first coiled-coil and PDZ domains are not necessary for these interactions, but deletion of the N-terminal portion of the leucine zipper abolishes dimerization. PIST may function as a scaffolding protein to link TC10 to signaling pathways.

Asunto(s)

Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas de la Membrana , Proteínas de Unión al GTP rho/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Unión Competitiva , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Proteínas Portadoras/genética , Línea Celular , Dimerización , Perfilación de la Expresión Génica , Proteínas de la Matriz de Golgi , Humanos , Leucina Zippers/genética , Proteínas de Transporte de Membrana , Ratones , Datos de Secuencia Molecular , Mutación/genética , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Especificidad por Sustrato , Transfección , Técnicas del Sistema de Dos Híbridos , Proteínas de Unión al GTP rho/química , Proteínas de Unión al GTP rho/genética

RESUMEN

Asymmetric cell division requires the orientation of mitotic spindles along the cell-polarity axis. In Drosophila neuroblasts, this involves the interaction of the proteins Inscuteable (Insc) and Partner of inscuteable (Pins). We report here that a human Pins-related protein, called LGN, is instead essential for the assembly and organization of the mitotic spindle. LGN is cytoplasmic in interphase cells, but associates with the spindle poles during mitosis. Ectopic expression of LGN disrupts spindle-pole organization and chromosome segregation. Silencing of LGN expression by RNA interference also disrupts spindle-pole organization and prevents normal chromosome segregation. We found that LGN binds the nuclear mitotic apparatus protein NuMA, which tethers spindles at the poles, and that this interaction is required for the LGN phenotype. Anti-LGN antibodies and the LGN-binding domain of NuMA both trigger microtubule aster formation in mitotic Xenopus egg extracts, and the NuMA-binding domain of LGN blocks aster assembly in egg extracts treated with taxol. Thus, we have identified a mammalian Pins homologue as a key regulator of spindle organization during mitosis.

Asunto(s)

Proteínas de Ciclo Celular , Proteínas de Drosophila , Proteínas de Insectos/metabolismo , Proteínas Nucleares/metabolismo , Huso Acromático/metabolismo , Proteínas de Xenopus , Animales , Anticuerpos , Antígenos Nucleares , Sitios de Unión/fisiología , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Expresión Génica/fisiología , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Riñón/citología , Mamíferos , Microtúbulos/metabolismo , Mitosis/fisiología , Proteínas Asociadas a Matriz Nuclear , Proteínas Nucleares/química , Oocitos/metabolismo , Unión Proteica/fisiología , ARN/farmacología , Conejos , Xenopus

RESUMEN

Importins are members of a family of transport receptors (karyopherins) that mediate the nucleocytoplasmic transport of protein and RNA cargoes. We identified importin-11 as a potential new human member of this family, on the basis of limited similarity to the Saccharomyces cerevisiae protein, Lph2p, and cloned the complete open reading frame. Importin-11 interacts with the Ran GTPase, and constitutively shuttles between the nuclear and cytoplasmic compartments. A yeast dihybrid screen identified UbcM2, an E2-type ubiquitin-conjugating enzyme, as a binding partner and potential transport cargo for importin-11. Importin-11 and UbcM2 interact directly, and the complex is disassembled by Ran:GTP but not by Ran:GDP. UbcM2 is constitutively nuclear and shuttles between the nuclear and cytoplasmic compartments. Nuclear import of UbcM2 requires Ran and importin-11, and is inhibited by wheatgerm agglutinin, energy depletion or dominant interfering mutants of Ran and importin-beta. These data establish importin-11 as a new member of the karyopherin family of transport receptors, and identify UbcM2 as a nuclear member of the E2 ubiquitin-conjugating enzyme family.

Asunto(s)

Proteínas Portadoras/metabolismo , Ligasas/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitinas/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Proteínas Portadoras/genética , Línea Celular , Permeabilidad de la Membrana Celular , Núcleo Celular/metabolismo , Clonación Molecular , Citoplasma/metabolismo , Técnica del Anticuerpo Fluorescente , Guanosina Trifosfato/metabolismo , Humanos , Carioferinas , Ratones , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/química , Proteínas Nucleares/genética , Filogenia , Unión Proteica , ARN Mensajero/análisis , ARN Mensajero/genética , Proteína de Unión al GTP ran/genética , Proteína de Unión al GTP ran/metabolismo

Asunto(s)

Proteínas de Unión al GTP rho/aislamiento & purificación , Clonación Molecular , Activación Enzimática , Vectores Genéticos , Proteínas Quinasas JNK Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación Puntual , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , Transducción de Señal , Técnicas del Sistema de Dos Híbridos , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismo

RESUMEN

PAR (partitioning-defective) proteins, which were first identified in the nematode Caenorhabditis elegans, are essential for asymmetric cell division and polarized growth, whereas Cdc42 mediates establishment of cell polarity. Here we describe an unexpected link between these two systems. We have identified a family of mammalian Par6 proteins that are similar to the C. elegans PDZ-domain protein PAR-6. Par6 forms a complex with Cdc42-GTP, with a human homologue of the multi-PDZ protein PAR-3 and with the regulatory domains of atypical protein kinase C (PKC) proteins. This assembly is implicated in the formation of normal tight junctions at epithelial cell-cell contacts. Thus, Par6 is a key adaptor that links Cdc42 and atypical PKCs to Par3.

Asunto(s)

Proteínas de Caenorhabditis elegans , Polaridad Celular , Proteínas del Helminto/metabolismo , Proteína Quinasa C/metabolismo , Proteínas/metabolismo , Uniones Estrechas/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Perros , Células Epiteliales/citología , Células Epiteliales/metabolismo , Técnica del Anticuerpo Fluorescente , Guanosina Trifosfato/metabolismo , Proteínas del Helminto/química , Proteínas del Helminto/genética , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Sustancias Macromoleculares , Proteínas de la Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Fosfoproteínas/metabolismo , Unión Proteica , Proteína Quinasa C/química , Proteínas Serina-Treonina Quinasas , Estructura Terciaria de Proteína , Proteínas/química , Proteínas/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Alineación de Secuencia , Especificidad por Sustrato , Uniones Estrechas/química , Técnicas del Sistema de Dos Híbridos , Proteína de la Zonula Occludens-1 , Proteínas de Unión al GTP rho/metabolismo

RESUMEN

RCC1, the only known guanine-nucleotide exchange factor for the Ran GTPase, is an approximately 45-kD nuclear protein that can bind chromatin. An important question concerns how RCC1 traverses the nuclear envelope. We now show that nuclear RCC1 is not exported readily in interphase cells and that the import of RCC1 into the nucleoplasm is extremely rapid. Import can proceed by at least two distinct mechanisms. The first is a classic import pathway mediated by basic residues within the NH(2)-terminal domain (NTD) of RCC1. This pathway is dependent upon both a preexisting Ran gradient and energy, and preferentially uses the importin-alpha3 isoform of importin-alpha. The second pathway is not mediated by the NTD of RCC1. This novel pathway does not require importin-alpha or importin-beta or the addition of any other soluble factor in vitro; however, this pathway is saturable and sensitive only to a subset of inhibitors of classical import pathways. Furthermore, the nuclear import of RCC1 does not require a preexisting Ran gradient or energy. We speculate that this second import pathway evolved to ensure that RCC1 never accumulates in the cytoplasm.

Asunto(s)

Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Intercambio de Guanina Nucleótido , Señales de Localización Nuclear , Proteínas Nucleares/metabolismo , Proteína de Unión al GTP ran/metabolismo , Secuencia de Aminoácidos , Transporte Biológico , Compartimento Celular , Permeabilidad de la Membrana Celular/efectos de los fármacos , Digitonina/farmacología , Células HeLa , Humanos , Modelos Biológicos , Datos de Secuencia Molecular

RESUMEN

The Ran binding protein RanBP1 is localized to the cytosol of interphase cells. A leucine-rich nuclear export signal (NES) near the C terminus of RanBP1 is essential to maintain this distribution. We now show that RanBP1 accumulates in nuclei of cells treated with the export inhibitor, leptomycin B, and collapse of the nucleocytoplasmic Ran:GTP gradient leads to equilibration of RanBP1 across the nuclear envelope. Low temperature prevents nuclear accumulation of RanBP1, suggesting that import does not occur via simple diffusion. Glutathione S-transferase (GST)-RanBP1(1-161), which lacks the NES, accumulates in the nucleus after cytoplasmic microinjection. In permeabilized cells, nuclear accumulation of GST-RanBP1(1-161) requires nuclear Ran:GTP but is not inhibited by a dominant interfering G19V mutant of Ran. Nuclear accumulation is enhanced by addition of exogenous karyopherins/importins or RCC1, both of which also enhance nuclear Ran accumulation. Import correlates with Ran concentration. Remarkably, an E37K mutant of RanBP1 does not import into the nuclei under any conditions tested despite the fact that it can form a ternary complex with Ran and importin beta. These data indicate that RanBP1 translocates through the pores by an active, nonclassical mechanism and requires Ran:GTP for nuclear accumulation. Shuttling of RanBP1 may function to clear nuclear pores of Ran:GTP, to prevent premature release of import cargo from transport receptors.

Asunto(s)

Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Factores de Intercambio de Guanina Nucleótido , Proteínas Nucleares/metabolismo , Receptores Citoplasmáticos y Nucleares , Proteína de Unión al GTP ran/metabolismo , Transporte Biológico Activo/efectos de los fármacos , Proteínas Portadoras/metabolismo , Compartimento Celular , Frío , Proteínas de Unión al ADN/metabolismo , Metabolismo Energético , Ácidos Grasos Insaturados/farmacología , Proteínas Fluorescentes Verdes , Guanosina Trifosfato/metabolismo , Carioferinas , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/aislamiento & purificación , Unión Proteica , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteína de Unión al GTP ran/genética , Proteína de Unión al GTP ran/aislamiento & purificación , Proteína Exportina 1

RESUMEN

Ran GTPase is required for nucleocytoplasmic transport of many types of cargo. Several proteins that recognize Ran in its GTP-bound state (Ran x GTP) possess a conserved Ran-binding domain (RanBD). Ran-binding protein-1 (RanBP1) has a single RanBD and is required for RanGAP-mediated GTP hydrolysis and release of Ran from nuclear transport receptors (karyopherins). In budding yeast (Saccharomyces cerevisiae), RanBP1 is encoded by the essential YRB1 gene; expression of mouse RanBP1 cDNA rescues the lethality of Yrb1-deficient cells. We generated libraries of mouse RanBP1 mutants and examined 11 mutants in vitro and for their ability to complement a temperature-sensitive yrb1 mutant (yrb1-51(ts)) in vivo. In 9 of the mutants, the alteration was a change in a residue (or 2 residues) that is conserved in all known RanBDs. However, 4 of these 9 mutants displayed biochemical properties indistinguishable from that of wild-type RanBP1. These mutants bound to Ran x GTP, stimulated RanGAP, inhibited the exchange activity of RCC1, and rescued growth of the yrb1-51(ts) yeast cells. Two of the 9 mutants altered in residues thought to be essential for interaction with Ran were unable to rescue growth of the yrb1(ts) mutant and did not bind detectably to Ran in vitro. However, one of these 2 mutants (and 2 others that were crippled in other RanBP1 functions) retained some ability to co-activate RanGAP. A truncated form of RanBP1 (lacking its nuclear export signal) was able to complement the yrb1(ts) mutation. When driven from the YRB1 promoter, 4 of the 5 mutants most impaired for Ran binding were unable to rescue growth of the yrb1(ts) cells; remarkably, these mutants could nevertheless form ternary complexes with importin-5 or importin-beta and Ran-GTP. The same mutants stimulated only inefficiently RanGAP-mediated GTP hydrolysis of the Ran x GTP x importin-5 complex. Thus, the essential biological activity of RanBP1 in budding yeast correlates not with Ran x GTP binding per se or with the ability to form ternary complexes with karyopherins, but with the capacity to potentiate RanGAP activity toward GTP-bound Ran in these complexes.

Asunto(s)

Proteínas de Ciclo Celular , Proteínas Fúngicas/genética , Factores de Intercambio de Guanina Nucleótido , Proteínas Nucleares/genética , Proteína de Unión al GTP ran/genética , Proteína de Unión al GTP ran/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Prueba de Complementación Genética , Carioferinas , Ratones , Modelos Moleculares , Mutagénesis , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Unión Proteica/genética , Saccharomyces cerevisiae/genética

RESUMEN

The full range of sequences that constitute nuclear localization signals (NLSs) remains to be established. Even though the sequence of the classical NLS contains polybasic residues that are recognized by importin-alpha, this import receptor can also bind cargo that contains no recognizable signal, such as STAT1. The situation is further complicated by the existence of six mammalian importin-alpha family members. We report the identification of an unusual type of NLS in human Ran binding protein 3 (RanBP3) that binds preferentially to importin-alpha3. RanBP3 contains a variant Ran binding domain most similar to that found in the yeast protein Yrb2p. Anti-RanBP3 immunofluorescence is predominantly nuclear. Microinjection of glutathione S-transferase-green fluorescent protein-RanBP3 fusions demonstrated that a region at the N terminus is essential and sufficient for nuclear localization. Deletion analysis further mapped the signal sequence to residues 40 to 57. This signal resembles the NLSs of c-Myc and Pho4p. However, several residues essential for import via the c-Myc NLS are unnecessary in the RanBP3 NLS. RanBP3 NLS-mediated import was blocked by competitive inhibitors of importin-alpha or importin-beta or by the absence of importin-alpha. Binding assays using recombinant importin-alpha1, -alpha3, -alpha4, -alpha5, and -alpha7 revealed a preferential interaction of the RanBP3 NLS with importin-alpha3 and -alpha4, in contrast to the simian virus 40 T-antigen NLS, which interacted to similar extents with all of the isoforms. Nuclear import of the RanBP3 NLS was most efficient in the presence of importin-alpha3. These results demonstrate that members of the importin-alpha family possess distinct preferences for certain NLS sequences and that the NLS consensus sequence is broader than was hitherto suspected.

Asunto(s)

Proteínas Portadoras/metabolismo , Señales de Localización Nuclear , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático , Animales , Sitios de Unión , Proteínas Portadoras/genética , Línea Celular , Núcleo Celular/metabolismo , Cricetinae , Humanos , Carioferinas , Proteínas Nucleares/genética , Isoformas de Proteínas

RESUMEN

Rabphilin is a protein that associates with the GTP-bound form of Rab3, a small GTPase that controls a late step in Ca(2+)-triggered exocytosis. Rabphilin is found only in neuroendocrine cells where it co-localises with Rab3A on the secretory vesicle membrane. The Rab3 binding domain (residues 45 to 170), located in the N-terminal part of Rabphilin, includes a cysteine-rich region with two zinc finger motifs that are required for efficient interaction with the small GTPase. To determine whether binding to Rab3A is necessary for the subcellular localisation of Rabphilin, we synthesised point mutants within the Rab3-binding domain. We found that two unique mutations (V61A and L83A) within an amphipathic alpha-helix of this region abolish detectable binding to endogenous Rab3, but only partially impair the targetting of the protein to secretory vesicles in PC12 and pancreatic HIT-T15 cells. Furthermore, both mutants transfected in the HIT-T15 beta cell line stimulate Ca(2+)-regulated exocytosis to the same extent as wild-type Rabphilin. Surprisingly, another Rabphilin mutant, R60A, which possesses a wild-type affinity for Rab3, and targets efficiently to membranes, does not potentiate regulated secretion. High affinity binding to Rab3 is therefore dispensable for the targetting of Rabphilin to secretory vesicles and for the potentiation of Ca(2+)-regulated secretion. The effects of Rabphilin on secretion may be mediated through interaction with another, unknown, factor that recognizes the Rab3 binding domain.

Asunto(s)

Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab3/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Gránulos Citoplasmáticos/metabolismo , GTP Fosfohidrolasas/metabolismo , Membranas Intracelulares/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Células PC12 , Mutación Puntual , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transfección , Proteínas de Transporte Vesicular , Proteínas de Unión al GTP rab/química , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab3/química , Rabfilina-3A