RESUMEN

PURPOSE: To evaluate histologic signs of toxicity of the protein tyrosine kinase inhibitor, imatinib mesylate, in rabbit eyes. METHODS: Twenty Dutch-belted rabbits underwent intravitreal injections of 0.1 ml solutions of imatinib mesylate. Ten rabbits were killed and enucleated 1 week after injection of imatinib mesylate (1.65 mg (four eyes), 165 microg (four eyes), and 16.5 microg (two eyes)). Ten rabbits injected with imatinib mesylate (165 microg (five eyes) and 825 microg (five eyes)) were enucleated 1 month later. Eyes were fixed in 10% formalin and stained with haematoxylin and eosin for microscopic examination. RESULTS: All four eyes injected with 1.65 mg of imatinib mesylate and enucleated at 1 week demonstrated ocular toxicity. All four eyes injected with 165 microg and enucleated at 1 week showed no ocular toxicity. One of the two eyes injected with 16.5 microg and enucleated at 1 week revealed focal areas of subretinal fluid and retinal undulations, suggestive of retinal oedema. None of the 10 eyes injected with imatinib mesylate at either the 165 or 825 microg dose and enucleated at 1 month showed ocular toxicity. CONCLUSIONS: Imatinib mesylate at 1.65 mg caused extensive retinal toxicity in rabbit eyes. In contrast, lower doses did not appear to cause toxicity, but may be associated with retinal oedema.

Asunto(s)

Nervio Óptico/efectos de los fármacos , Piperazinas/toxicidad , Inhibidores de Proteínas Quinasas/toxicidad , Pirimidinas/toxicidad , Retina/efectos de los fármacos , Animales , Benzamidas , Relación Dosis-Respuesta a Droga , Mesilato de Imatinib , Modelos Animales , Necrosis/patología , Nervio Óptico/patología , Conejos , Retina/patología , Cuerpo Vítreo

RESUMEN

Epithelial cells in vivo form tight cell-cell associations that spatially separate distinct apical and basolateral domains. These domains provide discrete cellular processes essential for proper tissue and organ development. Using confocal imaging and selective plasma membrane domain activation, the type I and type II transforming growth factor-beta (TGFbeta) receptors were found to be localized specifically at the basolateral surfaces of polarized Madin-Darby canine kidney (MDCK) cells. Receptors concentrated predominantly at the lateral sites of cell-cell contact, adjacent to the gap junctional complex. Cytoplasmic domain truncations for each receptor resulted in the loss of specific lateral domain targeting and dispersion to both the apical and basal domains. Whereas receptors concentrate basolaterally in regions of direct cell-cell contact in nonpolarized MDCK cell monolayers, receptor staining was absent from areas of noncell contact. In contrast to the defined basolateral polarity observed for the TGFbeta receptor complex, TGFbeta ligand secretion was found to be from the apical surfaces. Confocal imaging of MDCK cells with an antibody to TGFbeta1 confirmed a predominant apical localization, with a stark absence at the basal membrane. These findings indicate that cell adhesion regulates the localization of TGFbeta receptors in polarized epithelial cultures and that the response to TGFbeta is dependent upon the spatial distribution and secretion of TGFbeta receptors and ligand, respectively.

Asunto(s)

Polaridad Celular , Células Epiteliales/citología , Células Epiteliales/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Uniones Adherentes/metabolismo , Animales , Adhesión Celular , Línea Celular , Membrana Celular/metabolismo , Polaridad Celular/efectos de los fármacos , Perros , Humanos , Ligandos , Transporte de Proteínas , Receptores de Factores de Crecimiento Transformadores beta/química , Receptores de Factores de Crecimiento Transformadores beta/genética , Eliminación de Secuencia/genética , Transfección , Factor de Crecimiento Transformador beta/farmacología

RESUMEN

Transforming growth factor beta (TGF-beta) coordinates a number of biological events important in normal and pathophysiological growth. In this study, deletion and substitution mutations were used to identify receptor motifs modulating TGF-beta receptor activity. Initial experiments indicated that a COOH-terminal sequence between amino acids 482-491 in the kinase domain of the type I receptor was required for ligand-induced receptor signaling and down-regulation. These 10 amino acids are highly conserved in mammalian, Xenopus, and Drosophila type I receptors. Although mutation or deletion of the region (referred to as the NANDOR BOX, for nonactivating non-down-regulating) abolishes TGF-beta-dependent mitogenesis, transcriptional activity, type I receptor phosphorylation, and down-regulation in mesenchymal cultures, adjacent mutations also within the kinase domain are without effect. Moreover, a kinase-defective type I receptor can functionally complement a mutant BOX expressing type I receptor, documenting that when the BOX mutant is activated, it has kinase activity. These results indicate that the sequence between 482 and 491 in the type I receptor provides a critical function regulating activation of the TGF-beta receptor complex.

Asunto(s)

Endocitosis , Receptores de Factores de Crecimiento Transformadores beta/química , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Línea Celular , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Fibroblastos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Inhibidor 1 de Activador Plasminogénico/metabolismo , Estructura Terciaria de Proteína , Receptores de Factores de Crecimiento Transformadores beta/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Proteína Smad2 , Factores de Tiempo , Transactivadores/metabolismo

RESUMEN

Transforming growth factor-betas (TGF-beta) are multifunctional proteins capable of either stimulating or inhibiting mitosis, depending on the cell type. These diverse cellular responses are caused by stimulating a single receptor complex composed of type I and type II receptors. Using a chimeric receptor model where the granulocyte/monocyte colony-stimulating factor receptor ligand binding domains are fused to the transmembrane and cytoplasmic signaling domains of the TGF-beta type I and II receptors, we wished to describe the role(s) of specific amino acid residues in regulating ligand-mediated endocytosis and signaling in fibroblasts and epithelial cells. Specific point mutations were introduced at Y182, T200, and Y249 of the type I receptor and K277 and P525 of the type II receptor. Mutation of either Y182 or Y249, residues within two putative consensus tyrosine-based internalization motifs, had no effect on endocytosis or signaling. This is in contrast to mutation of T200 to valine, which resulted in ablation of signaling in both cell types, while only abolishing receptor down-regulation in fibroblasts. Moreover, in the absence of ligand, both fibroblasts and epithelial cells constitutively internalize and recycle the TGF-beta receptor complex back to the plasma membrane. The data indicate fundamental differences between mesenchymal and epithelial cells in endocytic sorting and suggest that ligand binding diverts heteromeric receptors from the default recycling pool to a pathway mediating receptor down-regulation and signaling.

Asunto(s)

Receptores de Activinas Tipo I , Endocitosis/fisiología , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Células 3T3 , Secuencias de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN/genética , Células Epiteliales/metabolismo , Fibroblastos/metabolismo , Humanos , Ratones , Modelos Biológicos , Mutagénesis Sitio-Dirigida , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/química , Receptores de Factores de Crecimiento Transformadores beta/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Tirosina/química

RESUMEN

Pneumocystis carinii is an opportunistic fungal pathogen phylogenetically related to the fission yeast Schizosaccharomyces pombe. P. carinii causes severe pneumonia in immunocompromised patients with AIDS and malignancies. Although the life cycle of P. carinii remains poorly characterized, morphologic studies of infected lung tissue indicate that P. carinii alternates between numerous small trophic forms and fewer large cystic forms. To understand further the molecular mechanisms that regulate progression of the cell cycle of P. carinii, we have sought to identify and characterize genes in P. carinii that are important regulators of eukaryotic cell cycle progression. In this study, we have isolated a cDNA from P. carinii that exhibits significant homology, but unique functional characteristics, to the mitotic phosphatase Cdc25 found in S. pombe. P. carinii Cdc25 was shown to rescue growth of the temperature-sensitive S. pombe cdc25-22 strain and thus provides an additional tool to investigate the unique P. carinii life cycle. Although P. carinii Cdc25 could also restore the DNA damage checkpoint in cdc25-22 cells, it was unable to restore fully the DNA replication checkpoint. The dissociation of checkpoint control at the level of Cdc25 indicates that Cdc25 may be under distinct regulatory control in mediating checkpoint signaling.

Asunto(s)

Ciclo Celular , Mitosis , Pneumocystis/citología , Pneumocystis/enzimología , Fosfatasas cdc25/metabolismo , Secuencia de Aminoácidos , Animales , Ciclo Celular/efectos de la radiación , Clonación Molecular , Daño del ADN/genética , Daño del ADN/efectos de la radiación , Replicación del ADN/efectos de la radiación , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Prueba de Complementación Genética , Cinética , Mitosis/efectos de la radiación , Datos de Secuencia Molecular , Mutación , Pneumocystis/genética , Pneumocystis/crecimiento & desarrollo , ARN de Hongos/análisis , ARN de Hongos/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Ratas , Saccharomyces/enzimología , Saccharomyces/genética , Alineación de Secuencia , Temperatura , Fosfatasas cdc25/química , Fosfatasas cdc25/genética

RESUMEN

Ligand binding to plasma membrane receptors initiates a series of events culminating in a variety of changes in cellular phenotypes. Although numerous publications have documented the activation/inactivation of signalling molecules following receptor binding, relatively few investigations have focused on the cellular compartment responsible for either initiating or selecting the particular pathway that mediates the response. Specifically, does receptor signalling occur only at the plasma membrane; is signalling dependent upon the location of defined endosome populations; or are components of both plasma membrane and endosomal activity operative depending upon the particular signalling pathway or cell type? This review addresses aspects of these questions by discussing the evidence supporting or contrasting the interplay between the endocytic and signalling systems for a subset of tyrosine kinase, serine/threonine kinase and G-protein-coupled receptors.

Asunto(s)

Sustancias de Crecimiento/fisiología , Receptores de Factores de Crecimiento/fisiología , Transducción de Señal/fisiología , Animales , Humanos

RESUMEN

Pneumocystis carinii causes severe pneumonia in immunocompromised patients. Recent studies indicate that P. carinii uses a Cdc2 cyclin-dependent kinase to control its proliferation. To further study the regulation of the life cycle of P. carinii, we characterized the P. carinii B-type cyclin termed Cdc13, whose binding to Cdc2 is necessary for kinase activity. Antibodies to B-type cyclins (Cdc13) specifically immunoprecipitated Cdc2/ Cdc13 complexes with associated kinase activity from P. carinii extracts. To clone P. carinii cdc13, degenerate polymerase chain reaction was undertaken using primers generated from amino-acid motifs conserved in fungal Cdc13 proteins. This amplicon was used to obtain full-length genomic and complementary DNA (cDNA) clones. A specific synthetic peptide antibody generated to P. carinii Cdc13 further demonstrated differential Cdc2/Cdc13 activity over the life cycle of P. carinii, with greater activity in cysts compared with trophic forms of the organism. Finally, P. carinii cdc13 cDNA was used to rescue mutant Schizosaccharomyces pombe strains containing temperature-sensitive deficiencies of endogenous Cdc13 activity, thus verifying function of the P. carinii Cdc13 protein. Therefore, P. carinii contains a Cdc13 cyclin, which is variably active over its life cycle and which promotes fungal proliferation.

Asunto(s)

Ciclina B/genética , Ciclina B/metabolismo , Pneumocystis/crecimiento & desarrollo , Neumonía por Pneumocystis/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Antifúngicos , Secuencia de Bases , División Celular/fisiología , Ciclina B/inmunología , ADN Complementario , Regulación de la Expresión Génica/fisiología , Genes Fúngicos/fisiología , Huésped Inmunocomprometido , Datos de Secuencia Molecular , Mutación , Pneumocystis/citología , Pneumocystis/enzimología , Proteínas Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Schizosaccharomyces , Temperatura

Asunto(s)

Factor de Crecimiento Transformador beta/fisiología , Animales , División Celular , Inhibidores de Crecimiento/fisiología , Humanos

RESUMEN

Transforming growth factor-beta (TGF-beta) family polypeptides regulate cell growth and differentiation by binding to single pass serine/threonine kinases referred to as TGF-beta type I and II receptors. Although interaction screens have shown that the immunophilin FKBP12 interacts with TGF-beta type I receptors, the role of FKBP12 in TGF-beta receptor action is presently unclear. Using a chimeric TGF-beta receptor system, we have shown a specific enhancement of internalization when FKBP12 binding to the type I receptor was prevented with rapamycin. Moreover, although earlier studies demonstrated that type II receptor kinase activity was required for optimal internalization in mesenchymal cells, we found that rapamycin functioned downstream of the type II receptor kinase. Thus, rather than modulating TGF-beta signaling, our data suggest a novel role for FKBP12 as a negative regulator of TGF-beta receptor endocytosis.

Asunto(s)

Inmunofilinas/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Endocitosis , Fibroblastos/metabolismo , Fibronectinas/biosíntesis , Fibronectinas/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Inmunofilinas/genética , Inmunosupresores/farmacología , Ratones , Mutagénesis Sitio-Dirigida , Inhibidor 1 de Activador Plasminogénico/metabolismo , Proteínas Serina-Treonina Quinasas , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Sirolimus/farmacología , Proteínas de Unión a Tacrolimus , Factores de Tiempo

Asunto(s)

Pneumocystis/enzimología , Fosfatasas cdc25/metabolismo , Animales , ADN de Hongos/genética , Pneumocystis/genética , Pneumocystis/crecimiento & desarrollo , Neumonía por Pneumocystis/microbiología , Reacción en Cadena de la Polimerasa/métodos , Ratas , Ratas Sprague-Dawley , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Temperatura , Fosfatasas cdc25/genética

RESUMEN

Pneumocystis carinii is an ascomycete phylogenetically related to Schizosaccharomyces pombe. Little is known about gene regulation in P. carinii. The removal of introns from pre-mRNA requires spliceosomal recognition of the intron-exon boundary. In S. pombe and higher eukaryotes, this boundary and a branch site within the intron are conserved. We recently demonstrated that P. carinii cdc2 cDNA can complement S. pombe containing conditional mutations of cdc2, an essential gene involved in cell cycle regulation. We next tested whether P. carinii genomic cdc2 (with six introns) could also complement S. pombe cdc2 mutants and found genomic sequences incapable of this activity. Reverse transcriptase PCR confirmed the inability of the S. pombe cdc2 mutants to splice the P. carinii genomic cdc2. Analysis of 83 introns from 19 P. carinii protein-encoding genes demonstrated that the sequence GTWWDW functions as a donor consensus in P. carinii, whereas YAG serves as an acceptor consensus. These sequences are similar in S. pombe; however, a branch site sequence was not found in the P. carinii genes studied.

Asunto(s)

Intrones , Pneumocystis/genética , Prueba de Complementación Genética , Empalme del ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Schizosaccharomyces/genética , Transformación Genética

RESUMEN

Transforming growth factor-beta (TGF-beta) induces distinct responses dependent upon the cellular context. It is unclear whether the initial receptor interactions identified in one cell type will be operative in another. Utilizing a chimeric receptor strategy we have examined the signaling and endocytic activity of both heteromeric (type I/type II) and homomeric (type I/type I or type II/type II) TGF-betaR interactions in Mv1Lu epithelial cells. In agreement with that observed in mesenchymal cells, all TGF-betaR signaling in Mv1Lu cells required the formation of a heteromeric type I-type II receptor complex. However, the initial endocytic response to TGF-betaR oligomerization was distinctly regulated in the two cell types. While heteromeric TGF-beta receptors were internalized and down-regulated, homomeric TGF-betaR interactions showed diminished endocytic activity in Mv1Lu cells. This contrasts to that observed in mesenchymal cultures where ligand bound to TGF-betaR homomers was internalized, yet the receptors were not down-regulated. Moreover, while previous reports have suggested that mutations at serine 172 or threonine 176 in the type I TGF-betaR separated transcriptional from proliferative responses, we found no separation of pathways or effect on initial endocytic activity when the analogous mutations were made in the chimeric receptors.

Asunto(s)

Receptores de Activinas Tipo I , Endocitosis/fisiología , Células Epiteliales/fisiología , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/fisiología , Receptores de Factores de Crecimiento Transformadores beta/química , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Transducción de Señal/fisiología , Sustitución de Aminoácidos , Animales , División Celular , Línea Celular , Membrana Celular/fisiología , Proteínas de la Matriz Extracelular/biosíntesis , Cinética , Pulmón , Sustancias Macromoleculares , Visón , Mutagénesis Sitio-Dirigida , Mutación Puntual , Multimerización de Proteína , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptor Tipo II de Factor de Crecimiento Transformador beta , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Serina , Treonina , Transfección , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta/fisiología

RESUMEN

Transforming growth factor beta (TGFbeta) superfamily polypeptides regulate cell growth and differentiation by binding to single pass serine/threonine kinases referred to as TGFbeta type I and type II receptors. Signal propagation is dependent upon heteromeric (type I-type II) complex formation and transphosphorylation of the type I receptor by the type II receptor. While many of the phosphorylation events necessary for receptor signaling have recently been characterized, the role of TGFbeta receptor kinase activity in modulating receptor endocytosis has not been addressed. To that end, we have used chimeric receptors consisting of the extracellular domain of the granulocyte/macrophage colony-stimulating factor alpha and beta receptors spliced to the TGFbeta type I and type II transmembrane and cytoplasmic domains to address the specific role of type I and/or type II receptor kinase activity in TGFbeta receptor internalization, down-regulation, and signaling. To inactivate chimeric receptor kinase activity, point mutations in the ATP binding site were made at amino acids 232 and 277 in the type I and type II receptor, respectively. Either of these mutations abolished plasminogen activator inhibitor 1 protein expression stimulated by granulocyte/macrophage colony-stimulating factor activation of chimeric heteromeric type I-type II TGFbeta receptors. They did not, however, modulate TGFbeta signaling stimulated through the endogenous TGFbeta receptor. Although TGFbeta receptor signaling was dependent upon the kinase activity of both chimeric receptors, the initial endocytic response was distinctly regulated by type I and/or type II receptor kinase activity. For instance, while heteromeric receptor complexes containing a kinase-inactive type I receptor were endocytosed similarly to wild type complexes, the kinase activity of the type II TGFbeta receptor was necessary for optimal internalization and receptor down-regulation. Furthermore, these responses were shown to occur independently of type II receptor autophosphorylation but require a type II receptor capable of transphosphorylation.

Asunto(s)

Receptores de Activinas Tipo I , Endocitosis/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Animales , Transporte Biológico , Clatrina/metabolismo , Células Clonales , Regulación hacia Abajo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Ligandos , Mesodermo/fisiología , Ratones , Modelos Biológicos , Mutagénesis Sitio-Dirigida , Fosforilación , Inhibidor 1 de Activador Plasminogénico/biosíntesis , Proteínas Serina-Treonina Quinasas/genética , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal

RESUMEN

The pathogenic fungus Pneumocystis carinii causes severe pneumonia in patients with impaired immunity, particularly patients with acquired immunodeficiency syndrome. The life cycle of P. carinii is poorly understood, and the inability to continuously culture P. carinii is a major limitation in understanding its cell biology. In fungi homologous to P. carinii, pheromone mating factors signal through a mitogen-activated protein kinase (MAPK) signal transduction cascade, resulting in mitotic cell cycle arrest and entry into a pathway of conjugation, cellular differentiation, and proliferation. Using degenerate PCR and library screening, we have identified a MAPK cDNA in P. carinii that is highly homologous to fungal MAPKs involved in the pheromone mating signal transduction cascade, and we demonstrate MAPK activity in P. carinii lysates with a specific antiserum derived from the translated P. carinii MAPK cDNA sequence.

Asunto(s)

Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Mapeo Cromosómico , Pneumocystis/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Cromosomas Fúngicos , Clonación Molecular , Hongos/enzimología , Biblioteca de Genes , Humanos , Cinética , Datos de Secuencia Molecular , Pneumocystis/genética , Reacción en Cadena de la Polimerasa , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido

RESUMEN

Pneumocystis carinii remains an important cause of pneumonia in patients with AIDS. Attachment of the organism to epithelial cells is a central event in establishing infection, impairing the growth potential of lung epithelial cells and thereby slowing repair. In light of investigations documenting a central role for cyclin-dependent kinases in controlling the cell cycle, we addressed the hypothesis that P. carinii inhibits epithelial cell growth by interfering with host epithelial cyclin-dependent kinase (cdk) activity. We observed that P. carinii significantly impaired growth of cultured mink lung epithelial cells, with effects observed after 48-72 h of treatment. However, the kinase activity associated with p34cdc2 or p33cdk2 was maximally inhibited as early as 24 h after P. carinii exposure. The inhibitory effect on cyclin-dependent kinase activity was mediated by the trophozoite form of P. carinii, in that highly purified trophozoites exerted marked inhibition of p34cdc2 activity. Growth impairment was similarly preceded by P. carinii-induced alteration in the state of epithelial cell p34cdc2 phosphorylation, with no change in p34cdc2 or p33cdk2 protein levels. These data strongly suggest that the antiproliferative activity of P. carinii on respiratory epithelium is mediated in part through modulation of the host cell cycle machinery.

Asunto(s)

Quinasas CDC2-CDC28 , Quinasas Ciclina-Dependientes/metabolismo , Células Epiteliales/citología , Células Epiteliales/microbiología , Pulmón/enzimología , Neumonía por Pneumocystis/enzimología , Animales , Ciclo Celular , Células Cultivadas , Quinasa 2 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/inmunología , Células Epiteliales/metabolismo , Histonas/análisis , Histonas/inmunología , Immunoblotting , Pulmón/citología , Pulmón/microbiología , Fosforilación , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley

RESUMEN

Pneumocystis carinii causes life-threatening pneumonia in immunocompromised patients. The inability to culture P. carinii has hampered basic investigations of the organism's life cycle, limiting the development of new therapies directed against it. Recent investigations indicate that P. carinii is a fungus phylogenetically related to other ascomycetes such as Schizosaccharomyces pombe. The cell cycles of S. pombe and homologous fungi are carefully regulated by cell-division-cycle molecules (cdc), particularly cell-division-cycle 2 (Cdc2), a serine-threonine kinase with essential activity at the G1 restriction point and for entry into mitosis. Antibodies to the proline-serine-threonine-alanine-isoleucine-arginine (PSTAIR) amino-acid sequence conserved in Cdc2 proteins specifically precipitated, from P. carinii extracts, a molecule with kinase activity consistent with a Cdc2-like protein. Cdc2 molecules exhibit differential activity throughout the life cycle of the organisms in which they occur. In accord with this, the P. carinii Cdc2 showed greater specific activity in P. carinii trophic forms (trophozoites) than in spore-case forms (cysts). In addition, complete genomic and complementary DNA (cDNA) sequences of P. carinii Cdc2 were cloned and found to be most closely homologus to the corresponding sequences of other pathogenic fungi. The function of P. carinii cdc2 cDNA was further documented through its ability to complement the DNA of mutant strains of S. pombe with temperature-sensitive deficiencies in Cdc2 activity. The P. carinii cdc2 cDNA restored normal Cdc2 function in these mutant strains of S. pombe, and promoted fungal proliferation. These studies represent the first molecular analysis of the cell-cycle-regulatory machinery in P. carinii. Further understanding of P. carinii's life cycle promises novel insights for preventing and treating the intractable infection it causes in immunocompromised patients.

Asunto(s)

Proteína Quinasa CDC2/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Pneumocystis/genética , Secuencia de Aminoácidos , Secuencia de Bases , Ciclo Celular/fisiología , Clonación Molecular , ADN Complementario/genética , Células Eucariotas/enzimología , Regulación del Desarrollo de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Prueba de Complementación Genética , Biblioteca Genómica , Datos de Secuencia Molecular , Pneumocystis/citología , Pneumocystis/enzimología , ARN de Hongos/genética , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Homología de Secuencia de Aminoácido , Esporas Fúngicas/enzimología

RESUMEN

Transforming growth factor beta (TGF beta) family ligands initiate a cascade of events capable of modulating cellular growth and differentiation. The receptors responsible for transducing these cellular signals are referred to as the type I and type II TGF beta receptors. Ligand binding to the type II receptor results in the transphosphorylation and activation of the type I receptor. This heteromeric complex then propagates the signal(s) to downstream effectors. There is presently little data concerning the fate of TGF beta receptors after ligand binding, with conflicting reports indicating no change or decreasing cell surface receptor numbers. To address the fate of ligand-activated receptors, we have used our previously characterized chimeric receptors consisting of the ligand binding domain from the granulocyte/macrophage colony-stimulating factor alpha or beta receptor fused to the transmembrane and cytoplasmic domain of the type I or type II TGF beta receptor. This system not only provides the necessary sensitivity and specificity to address these types of questions but also permits the differentiation of endocytic responses to either homomeric or heteromeric intracellular TGF beta receptor oligomerization. Data are presented that show, within minutes of ligand binding, chimeric TGF beta receptors are internalized. However, although all the chimeric receptor combinations show similar internalization rates, receptor down-regulation occurs only after activation of heteromeric TGF beta receptors. These results indicate that effective receptor down-regulation requires cross-talk between the type I and type II TGF beta receptors and that TGF beta receptor heteromers and homomers show distinct trafficking behavior.

Asunto(s)

Receptores de Activinas Tipo I , Regulación hacia Abajo/fisiología , Endocitosis/fisiología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Clatrina/fisiología , Dimerización , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Ligandos , Potasio/fisiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal

Asunto(s)

Ciclo Celular/fisiología , Pneumocystis/metabolismo , Neumonía por Pneumocystis/microbiología , Alveolos Pulmonares/microbiología , Adhesión Bacteriana/fisiología , División Celular/fisiología , Línea Celular , Epitelio/microbiología , Humanos , Huésped Inmunocomprometido , Microscopía Electrónica , Pneumocystis/crecimiento & desarrollo , Neumonía por Pneumocystis/patología , Alveolos Pulmonares/patología

Asunto(s)

Proteína Quinasa CDC2/metabolismo , Ciclo Celular/fisiología , Pneumocystis/enzimología , Animales , Proteína Quinasa CDC2/genética , Clonación Molecular , ADN de Hongos/genética , Genes Fúngicos/genética , Prueba de Complementación Genética , Histonas/metabolismo , Mutación , Fragmentos de Péptidos , Fosforilación , Pneumocystis/genética , Ratas , Ratas Sprague-Dawley , Schizosaccharomyces/enzimología

RESUMEN

Transforming growth factor-beta (TGF-beta) belongs to a family of ligands that regulate cell growth and differentiation. The most commonly observed receptors are referred to as the type I, type II, and type III (betaglycan) TGF-beta receptors. Two receptor models have been presented to account for the various cellular responses to TGF-beta. The first proposes that all TGF-beta signaling results from the formation of a heteromeric type I/type II complex, while the second suggests that distinct type I or type II TGF-beta receptor combinations mediate aspects of TGF-beta signaling. We have addressed this general question relating to TGF-beta signaling by constructing chimeric receptors consisting of the extracellular domain of the granulocyte/macrophage colony-stimulating factor (GM-CSF) alpha or beta receptor fused to the transmembrane and cytoplasmic domain of the type I or type II TGF-beta receptor. Since high affinity GM-CSF binding requires dimerization of the alpha and beta ligand binding subunits, the response elicited by defined type I and/or type II TGF-beta receptor cytoplasmic domain homomers or heteromers can be examined. We show in mesenchymal AKR-2B cells that while TGF-beta-dependent transient luciferase activity, endogenous gene activity, and long-term biological responses are similarly induced by activating the chimeric heteromeric receptors with GM-CSF as the endogenous TGF-beta receptor, chimeric homomeric type I/type I or type II/type II receptors are signaling-incompetent.

Asunto(s)

Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/fisiología , Secuencia de Bases , Adhesión Celular , Línea Celular , Relación Dosis-Respuesta a Droga , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-Actividad