RESUMEN
A subset of DNA helicases, the RecQ family, has been found to be associated with the p53-mediated apoptotic pathway and is involved in maintaining genomic integrity. This family contains the BLM and WRN helicases, in which germline mutations are responsible for Bloom and Werner syndromes, respectively. TFIIH DNA helicases, XPB and XPD, are also components in this apoptotic pathway. We hypothesized that there may be some redundancy between helicases in their ability to complement the attenuated p53-mediated apoptotic levels seen in cells from individuals with diseases associated with these defective helicase genes. The attenuated apoptotic phenotype in Bloom syndrome cells was rescued not only by ectopic expression of BLM, but also by WRN or XPB, both 3' --> 5' helicases, but not expression of the 5' --> 3' helicase XPD. Overexpression of Sgs1, a WRN/BLM yeast homolog, corrected the reduction in BS cells only, which is consistent with Sgs1 being evolutionarily most homologous to BLM. A restoration of apoptotic levels in cells from WS, XPB or XPD patients was attained only by overexpression of the specific helicase. Our data suggest a limited redundancy in the pathways of these RecQ helicases in p53-induced apoptosis.
Asunto(s)
Apoptosis/fisiología , ADN Helicasas/metabolismo , Proteína p53 Supresora de Tumor/fisiología , Síndrome de Bloom/enzimología , Mutación de Línea Germinal , Humanos , Síndrome de Werner/enzimologíaRESUMEN
Werner syndrome (WS) is a human premature aging disorder characterized by chromosomal instability. The cellular defects of WS presumably reflect compromised or aberrant function of a DNA metabolic pathway that under normal circumstances confers stability to the genome. We report a novel interaction of the WRN gene product with the human 5' flap endonuclease/5'-3' exonuclease (FEN-1), a DNA structure-specific nuclease implicated in DNA replication, recombination and repair. WS protein (WRN) dramatically stimulates the rate of FEN-1 cleavage of a 5' flap DNA substrate. The WRN-FEN-1 functional interaction is independent of WRN catalytic function and mediated by a 144 amino acid domain of WRN that shares homology with RecQ DNA helicases. A physical interaction between WRN and FEN-1 is demonstrated by their co-immunoprecipitation from HeLa cell lysate and affinity pull-down experiments using a recombinant C-terminal fragment of WRN. The underlying defect of WS is discussed in light of the evidence for the interaction between WRN and FEN-1.
Asunto(s)
ADN Helicasas/fisiología , Endodesoxirribonucleasas/metabolismo , Síndrome de Werner/genética , Adenosina Trifosfatasas/fisiología , Catálisis , ADN/metabolismo , ADN Helicasas/química , Proteínas de Unión al ADN/fisiología , Endodesoxirribonucleasas/química , Activación Enzimática , Exodesoxirribonucleasas , Exonucleasas/fisiología , Endonucleasas de ADN Solapado , Células HeLa , Humanos , Sustancias Macromoleculares , Fragmentos de Péptidos/metabolismo , Antígeno Nuclear de Célula en Proliferación/fisiología , Estructura Terciaria de Proteína , RecQ Helicasas , Proteínas Recombinantes de Fusión/metabolismo , Proteína de Replicación A , Helicasa del Síndrome de WernerRESUMEN
Messenger RNAs are exported from the nucleus as large ribonucleoprotein complexes (mRNPs). To date, proteins implicated in this process include TAP/Mex67p and RAE1/Gle2p and are distinct from the nuclear transport receptors of the beta-related, Ran-binding protein family. Mex67p is essential for mRNA export in yeast. Its vertebrate homolog TAP has been implicated in the export of cellular mRNAs and of simian type D viral RNAs bearing the constitutive transport element (CTE). Here we show that TAP is predominantly localized in the nucleoplasm and at both the nucleoplasmic and cytoplasmic faces of the nuclear pore complex (NPC). TAP interacts with multiple components of the NPC including the nucleoporins CAN, Nup98, Nup153, p62, and with three major NPC subcomplexes. The nucleoporin-binding domain of TAP comprises residues 508-619. In HeLa cells, this domain is necessary and sufficient to target GFP-TAP fusions to the nuclear rim. Moreover, the isolated domain strongly competes multiple export pathways in vivo, probably by blocking binding sites on the NPC that are shared with other transport receptors. Microinjection experiments implicate this domain in the export of specific CTE-containing RNAs. Finally, we show that TAP interacts with transportin and with two proteins implicated in the export of cellular mRNAs: RAE1/hGle2 and E1B-AP5. The interaction of TAP with nucleoporins, its direct binding to the CTE RNA, and its association with two mRNP binding proteins suggest that TAP is an RNA export mediator that may bridge the interaction between specific RNP export substrates and the NPC.
Asunto(s)
Membrana Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Células HeLa , Ribonucleoproteínas Nucleares Heterogéneas , Humanos , Carioferinas , Proteínas de la Membrana/metabolismo , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Proteínas Nucleares/ultraestructura , Oocitos , Unión Proteica , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/ultraestructura , Receptores Citoplasmáticos y Nucleares/metabolismo , XenopusRESUMEN
Dbp5 is a DEAD-box protein essential for mRNA export from the nucleus in yeast. Here we report the isolation of a cDNA encoding human Dbp5 (hDbp5) which is 46% identical to yDbp5p. Like its yeast homologue, hDbp5 is localized within the cytoplasm and at the nuclear rim. By immunoelectron microscopy, the nuclear envelope-bound fraction of Dbp5 has been localized to the cytoplasmic fibrils of the nuclear pore complex (NPC). Consistent with this localization, we show that both the human and yeast proteins directly interact with an N-terminal region of the nucleoporins CAN/Nup159p. In a conditional yeast strain in which Nup159p is degraded when shifted to the nonpermissive temperature, yDbp5p dissociates from the NPC and localizes to the cytoplasm. Thus, Dbp5 is recruited to the NPC via a conserved interaction with CAN/Nup159p. To investigate its function, we generated defective hDbp5 mutants and analysed their effects in RNA export by microinjection in Xenopus oocytes. A mutant protein containing a Glu-->Gln change in the conserved DEAD-box inhibited the nuclear exit of mRNAs. Together, our data indicate that Dbp5 is a conserved RNA-dependent ATPase which is recruited to the cytoplasmic fibrils of the NPC where it participates in the export of mRNAs out of the nucleus.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Citoplasma/metabolismo , Proteínas de la Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático , ARN Helicasas , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfatasas/genética , Secuencia de Aminoácidos , Transporte Biológico , Clonación Molecular , Secuencia Conservada , ARN Helicasas DEAD-box , Evolución Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Homología de Secuencia de AminoácidoRESUMEN
The constitutive transport element (CTE) of the type D retroviruses promotes nuclear export of unspliced viral RNAs apparently by recruiting host factor(s) required for export of cellular messenger RNAs. Here, we report the identification of TAP as the cellular factor that specifically binds to wild-type CTE but not to export-deficient CTE mutants. Microinjection experiments performed in Xenopus oocytes demonstrate that TAP directly stimulates CTE-dependent export. Furthermore, TAP overcomes the mRNA export block caused by the presence of saturating amounts of CTE RNA. Thus, TAP, like its yeast homolog Mex67p, is a bona fide mRNA nuclear export mediator. TAP is the second cellular RNA binding protein shown to be directly involved in the export of its target RNA.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Núcleo Celular/metabolismo , Proteínas de Transporte Nucleocitoplasmático , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Animales , Transporte Biológico/fisiología , Clonación Molecular , Proteínas Fúngicas/metabolismo , Células HeLa , Humanos , Intrones/fisiología , Mutagénesis/fisiología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Conformación de Ácido Nucleico , Oocitos/metabolismo , ARN Mensajero/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Homología de Secuencia de Aminoácido , XenopusRESUMEN
p85/p110 phosphoinositide 3-kinase (PI3K) is a heterodimer composed of a p85-regulatory and a p110-catalytic subunit, which is involved in a variety of cellular responses including cytoskeletal organization, cell survival and proliferation. We describe here the cloning and characterization of p65-PI3K, a mutant of the regulatory subunit of PI3K, which includes the initial 571 residues of the wild type p85alpha-protein linked to a region conserved in the eph tyrosine kinase receptor family. We demonstrate that this mutation, obtained from a transformed cell, unlike previously engineered mutations of the regulatory subunit, induces the constitutive activation of PI3K and contributes to cellular transformation. This report links the PI3K enzyme to mammalian tumor development for the first time.
Asunto(s)
Proteínas de Unión al Calcio , Oncogenes/genética , Fosfatidilinositol 3-Quinasas/genética , Proteínas Serina-Treonina Quinasas , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Línea Celular Transformada , Transformación Celular Neoplásica/metabolismo , Clonación Molecular , Inducción Enzimática/genética , Inducción Enzimática/fisiología , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/fisiología , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/fisiología , Fosfatidilinositol 3-Quinasas/análisis , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositoles/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/fisiología , Secuencias Reguladoras de Ácidos Nucleicos/genética , Sinaptotagmina I , SinaptotagminasRESUMEN
The GTPase Ran is essential for nuclear import of proteins with a classical nuclear localization signal (NLS). Ran's nucleotide-bound state is determined by the chromatin-bound exchange factor RCC1 generating RanGTP in the nucleus and the cytoplasmic GTPase activating protein RanGAP1 depleting RanGTP from the cytoplasm. This predicts a steep RanGTP concentration gradient across the nuclear envelope. RanGTP binding to importin-beta has previously been shown to release importin-alpha from -beta during NLS import. We show that RanGTP also induces release of the M9 signal from the second identified import receptor, transportin. The role of RanGTP distribution is further studied using three methods to collapse the RanGTP gradient. Nuclear injection of either RanGAP1, the RanGTP binding protein RanBP1 or a Ran mutant that cannot stably bind GTP. These treatments block major export and import pathways across the nuclear envelope. Different export pathways exhibit distinct sensitivities to RanGTP depletion, but all are more readily inhibited than is import of either NLS or M9 proteins, indicating that the block of export is direct rather than a secondary consequence of import inhibition. Surprisingly, nuclear export of several substrates including importin-alpha and -beta, transportin, HIV Rev and tRNA appears to require nuclear RanGTP but may not require GTP hydrolysis by Ran, suggesting that the energy for their nuclear export is supplied by another source.
Asunto(s)
Transporte Biológico/fisiología , Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Proteínas Activadoras de GTPasa , Factores de Intercambio de Guanina Nucleótido , Proteínas Nucleares/fisiología , Animales , Proteínas Portadoras/metabolismo , Compartimento Celular , Citoplasma/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al GTP/metabolismo , Productos del Gen rev/metabolismo , Guanosina Trifosfato/metabolismo , Carioferinas , Sustancias Macromoleculares , Proteínas Nucleares/metabolismo , Oocitos , ARN/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteína de Unión al GTP ranRESUMEN
Growth hormone (GH) plays a significant role in normal growth and development. Signaling to the cell is believed to require growth hormone receptor (GHR) dimerization, which occurs following binding of a single growth hormone molecule to each of two receptors. We have developed human growth hormone receptor-specific monoclonal antibodies, one of which was used here to characterize hormone/receptor interactions. This antibody, GHR05, is directed against the hinge spanning subdomains I and II of the receptor's extracellular region. Antibody binding to the cell surface receptor increases upon receptor binding to growth hormone, but not when it binds a mutant form, hGHG120R, which does not trigger receptor activation. Growth hormone binding thus appears to lead to a conformational change in the receptor epitope recognized by GHR05, giving rise to the active dimer configuration, necessary for signal transduction. Using a chimeric receptor-expressing, growth hormone-dependent murine cell line, we find that GHR05 binds to the receptor in the absence of human GH and delivers a signal leading to cell proliferation. Finally, GHR05 treatment of IM-9 cells, a human cell line expressing a functional human GHR, leads to cell proliferation mediated by the generation of GH-specific signals, including phosphorylation of the JAK2 tyrosine kinase and activation of STAT5.
Asunto(s)
Hormona de Crecimiento Humana/metabolismo , Proteínas de la Leche , Proteínas Proto-Oncogénicas , Receptores de Somatotropina/química , Transducción de Señal , Animales , Anticuerpos Monoclonales , Ciclo Celular , División Celular , Proteínas de Unión al ADN/metabolismo , Epítopos/metabolismo , Humanos , Janus Quinasa 2 , Ratones , Modelos Biológicos , Conformación Proteica , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores de Somatotropina/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Factor de Transcripción STAT5 , Relación Estructura-Actividad , Transactivadores/metabolismoRESUMEN
Neuroendocrine hormones have long been thought to play a role in lymphoid development and function. In particular, growth hormone has been shown to mediate thymic development as well as to promote T cell engraftment in severe combined immunodeficiency mice. Murine T helper cells are classified into two subsets based on their cytokine production pattern. Here, we report that transgenic mice for bovine growth hormone show significant alterations in T cell function and decreased capability for cytokine production, an effect that is more acute in T helper cells as measured by their inability to produce IL-4 upon in vivo injection with Staphylococcus aureus enterotoxin B. Furthermore, upon immunization with conventional Ags, growth hormone transgenic mice produce an altered Ig isotype pattern characterized by a response shift from IgG1 in nontransgenic mice to IgG2 in transgenic mice. The impaired T cell responses correlated with survival from septic shock mediated by bacterial enterotoxins. We conclude that growth hormone may have the potential of regulating immune responses in pathologic processes associated with hyperactivation of T cells or with massive cytokine production.