RESUMEN
Our previous studies revealed that the expression of the 19-kDa protein prenylated Rab acceptor 1 domain family, member 2 (PRAF2) is elevated in cancer tissues of the breast, colon, lung, and ovary, when compared to noncancerous tissues of paired samples. PRAF2 mRNA expression also correlated with several genetic and clinical features and is a candidate prognostic marker in the pediatric cancer neuroblastoma. The PRAF2-related proteins, PRAF1 and PRAF3, play multiple roles in cellular processes, including endo/exocytic vesicle trafficking and glutamate uptake. PRAF2 shares a high sequence homology with these family members, but its function remains unknown. In this study, we examined PRAF2 mRNA and protein expression in 20 different human cancer types using Affymetrix microarray and human tissue microarray (TMA) analyses, respectively. In addition, we investigated the subcellular distribution of PRAF2 by immunofluorescence microscopy and cell fractionation studies. PRAF2 mRNA and protein expression was elevated in several cancer tissues with highest levels in malignant glioma. At the molecular level, we detected native PRAF2 in small, vesicle-like structures throughout the cytoplasm as well as in and around cell nuclei of U-87 malignant glioma cells. We further found that monomeric and dimeric forms of PRAF2 are associated with different cell compartments, suggesting possible functional differences. Importantly, PRAF2 down-regulation by RNA interference significantly reduced the cell viability, migration, and invasiveness of U-87 cells. This study shows that PRAF2 expression is elevated in various tumors with exceptionally high expression in malignant gliomas, and PRAF2 therefore presents a candidate molecular target for therapeutic intervention.
Asunto(s)
Neoplasias Encefálicas/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Glioma/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Astrocitoma/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Proteínas Portadoras/química , Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral , Movimiento Celular , Supervivencia Celular , Glioblastoma/genética , Glioma/metabolismo , Glioma/patología , Humanos , Proteínas de la Membrana/química , Oligodendroglioma/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Análisis por Matrices de Proteínas , Prenilación de Proteína , ARN Mensajero/genética , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Cyclic nucleotide-gated channels (CNGCs) in the plasma membrane transport K+ and other cations; however, their roles in the response and adaptation of plants to environmental salinity are unclear. Growth, cation contents, salt tolerance and K+ fluxes were assessed in wild-type and two AtCNGC10 antisense lines (A2 and A3) of Arabidopsis thaliana (L.) Heynh. Compared with the wild-type, mature plants of both antisense lines had altered K+ and Na+ concentrations in shoots and were more sensitive to salt stress, as assessed by biomass and Chl fluorescence. The shoots of A2 and A3 plants contained higher Na+ concentrations and significantly higher Na+/K+ ratios compared with wild-type, whereas roots contained higher K+ concentrations and lower Na+/K+ ratios. Four-day-old seedlings of both antisense lines exposed to salt stress had smaller Na+/K+ ratios and longer roots than the wild-type. Under sudden salt treatment, the Na+ efflux was higher and the K+ efflux was smaller in the antisense lines, indicating that AtCNGC10 might function as a channel providing Na+ influx and K+ efflux at the root/soil interface. We conclude that the AtCNGC10 channel is involved in Na+ and K+ transport during cation uptake in roots and in long-distance transport, such as phloem loading and/or xylem retrieval. Mature A2 and A3 plants became more salt sensitive than wild-type plants because of impaired photosynthesis induced by a higher Na+ concentration in the leaves.
Asunto(s)
Adaptación Fisiológica/efectos de los fármacos , Proteínas de Arabidopsis/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/fisiología , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Cloruro de Sodio/farmacología , Biomasa , Transporte Iónico/efectos de los fármacos , Meristema/efectos de los fármacos , Meristema/crecimiento & desarrollo , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/fisiología , Plantones/efectos de los fármacos , Plantones/metabolismoRESUMEN
BACKGROUND: The cyclic nucleotide-gated ion channels (CNGCs) maintain cation homeostasis essential for a wide range of physiological processes in plant cells. However, the precise subcellular locations and trafficking of these membrane proteins are poorly understood. This is further complicated by a general deficiency of information about targeting pathways of membrane proteins in plants. To investigate CNGC trafficking and localization, we have measured Atcngc5 and Atcngc10 expression in roots and leaves, analyzed AtCNGC10-GFP fusions transiently expressed in protoplasts, and conducted immunofluorescence labeling of protoplasts and immunoelectron microscopic analysis of high pressure frozen leaves and roots. RESULTS: AtCNGC10 mRNA and protein levels were 2.5-fold higher in roots than leaves, while AtCNGC5 mRNA and protein levels were nearly equal in these tissues. The AtCNGC10-EGFP fusion was targeted to the plasma membrane in leaf protoplasts, and lightly labeled several intracellular structures. Immunofluorescence microscopy with affinity purified CNGC-specific antisera indicated that AtCNGC5 and AtCNGC10 are present in the plasma membrane of protoplasts. Immunoelectron microscopy demonstrated that AtCNGC10 was associated with the plasma membrane of mesophyll, palisade parenchyma and epidermal cells of leaves, and the meristem, columella and cap cells of roots. AtCNCG10 was also observed in the endoplasmic reticulum and Golgi cisternae and vesicles of 50-150 nm in size. Patch clamp assays of an AtCNGC10-GFP fusion expressed in HEK293 cells measured significant cation currents. CONCLUSION: AtCNGC5 and AtCNGC10 are plasma membrane proteins. We postulate that AtCNGC10 traffics from the endoplasmic reticulum via the Golgi apparatus and associated vesicles to the plasma membrane. The presence of the cation channel, AtCNGC10, in root cap meristem cells, cell plate, and gravity-sensing columella cells, combined with the previously reported antisense phenotypes of decreased gravitropic and cell enlargement responses, suggest roles of AtCNGC10 in modulating cation balance required for root gravitropism, cell division and growth.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Canales Iónicos/metabolismo , Proteínas de Arabidopsis/análisis , Proteínas de Arabidopsis/fisiología , Línea Celular , Canales Catiónicos Regulados por Nucleótidos Cíclicos/análisis , Canales Catiónicos Regulados por Nucleótidos Cíclicos/fisiología , Retículo Endoplásmico/ultraestructura , Aparato de Golgi/ultraestructura , Proteínas Fluorescentes Verdes/análisis , Humanos , Canales Iónicos/análisis , Canales Iónicos/fisiología , Técnicas de Placa-Clamp , Hojas de la Planta/metabolismo , Raíces de Plantas/metabolismo , Transporte de Proteínas , Protoplastos/metabolismo , Protoplastos/ultraestructura , Proteínas Recombinantes de Fusión/análisisRESUMEN
S-adenosylmethionine decarboxylase (AdoMetDC) is an essential enzyme of polyamine (PA) biosynthesis, and both AdoMetDC and PA levels are often up-regulated in cancer cells. The second-generation inhibitor SAM486A inhibits AdoMetDC enzyme activity and has been evaluated in phase II clinical cancer trials. However, little is known about the mechanism of action and potential use of this therapeutic drug in the treatment of the pediatric cancer neuroblastoma (NB). Here, we show that p53 wild-type NB cells are highly sensitive to SAM486A treatment. Most notably, SAM486A treatment resulted in the rapid accumulation of proapoptotic proteins p53 and Mdm2. Concomitant with the increase of proteins at endogenous levels, the in vivo phosphorylation of p53 at residues Ser(46)/Ser(392) and Mdm2 at residue Ser(166) was observed. Moreover, the antiapoptotic protein Akt/protein kinase B was down-regulated and also dephosphorylated at residue Ser(473) in a dose- and time-dependent manner and NB cells entered apoptotic cell death. The results presented in this study highlight the importance of PA homeostasis and provide a direct link between PA metabolism and apoptotic cell signaling pathways in p53 wild-type NB cells. PA inhibitors such as SAM486A may be effective alternative agents for the treatment of NBs with or without MYCN amplification.
Asunto(s)
Adenosilmetionina Descarboxilasa/antagonistas & inhibidores , Amidinas/farmacología , Apoptosis/efectos de los fármacos , Indanos/farmacología , Neuroblastoma/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Antineoplásicos/farmacología , Western Blotting , Proliferación Celular/efectos de los fármacos , Citometría de Flujo , Humanos , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/metabolismo , Fosforilación/efectos de los fármacos , S-Adenosilmetionina/farmacologíaRESUMEN
Ornithine decarboxylase (ODC) is a key enzyme in mammalian polyamine biosynthesis that is up-regulated in various types of cancer. We previously showed that treating human neuroblastoma (NB) cells with the ODC inhibitor alpha-difluoromethylornithine (DFMO) depleted polyamine pools and induced G1 cell cycle arrest without causing apoptosis. However, the precise mechanism by which DFMO provokes these changes in NB cells remained unknown. Therefore, we further examined the effects of DFMO, alone and in combination with phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 or Akt/protein kinase B (PKB) inhibitor IV, on the regulation of cell survival and cell cycle-associated pathways in LAN-1 NB cells. In the present study, we found that the inhibition of ODC by DFMO promotes cell survival by inducing the phosphorylation of Akt/PKB at residue Ser473 and glycogen synthase kinase-3beta at Ser9. Intriguingly, DFMO also induced the phosphorylation of p27Kip1 at residues Ser10 (nuclear export) and Thr198 (protein stabilization) but not Thr187 (proteasomal degradation). The combined results from this study provide evidence for a direct cross-talk between ODC-dependent metabolic processes and well-established cell signaling pathways that are activated during NB tumorigenesis. The data suggest that inhibition of ODC by DFMO induces two opposing pathways in NB: one promoting cell survival by activating Akt/PKB via the PI3K/Akt pathway and one inducing p27Kip1/retinoblastoma-coupled G1 cell cycle arrest via a mechanism that regulates the phosphorylation and stabilization of p27Kip1. This study presents new information that may explain the moderate efficacy of DFMO monotherapy in clinical trials and reveals potential new targets for DFMO-based combination therapies for NB treatment.
Asunto(s)
Eflornitina/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/enzimología , Proteína Oncogénica v-akt/metabolismo , Inhibidores de la Ornitina Descarboxilasa , Poliaminas Biogénicas/antagonistas & inhibidores , Poliaminas Biogénicas/metabolismo , Línea Celular Tumoral , Cromonas/farmacología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Morfolinas/farmacología , Proteína Oncogénica v-akt/antagonistas & inhibidores , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Cyclic nucleotide gated channels (CNGCs) that are regulated by calmodulin (CaM) have been shown to play essential roles in signal transduction, metabolism, and growth in animals. By contrast, very little is known about the subcellular location and the function of these channels in plants. Here we report on the effects of antisense suppression of the expression of AtCNGC10, a putative K+ channel, and the immunolocalization of the protein using an AtCNGC10-specific antiserum. In Arabidopsis thaliana leaves, AtCNGC10 was localized to the plasma membrane of mesophyll and parenchyma cells. Antisense AtCNGC10 plants had 40% of the AtCNGC10 mRNA levels and virtually undetectable protein levels relative to wild type plants. Antisense expression of AtCNGC10 did not affect the mRNA levels of AtCNGC13, the most closely related CNGC family member in the genome. Relative to wild type Columbia, antisense AtCNGC10 plants flowered 10 days earlier, and had a 25% reduction in leaf surface area, thickness and palisade parenchyma cell length. Their roots responded more slowly to gravitropic changes and the chloroplasts accumulated more starch. We propose that AtCNGC10, through interactions with CaM and cGMP, modulates cellular K+ balance across the plasma membrane, and that perturbations of this K+ gradient affect numerous growth and developmental processes.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Calmodulina/metabolismo , Canales Iónicos/metabolismo , Hojas de la Planta/metabolismo , Almidón/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Cloroplastos/metabolismo , Cloroplastos/ultraestructura , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Regulación de la Expresión Génica de las Plantas , Immunoblotting , Canales Iónicos/genética , Microscopía Electrónica de Transmisión , Microscopía Inmunoelectrónica , Hojas de la Planta/ultraestructura , Unión Proteica , Factores de TiempoRESUMEN
We have isolated and characterised AtCNGC10, one of the 20 members of the family of cyclic nucleotide (CN)-gated and calmodulin (CaM)-regulated channels (CNGCs) from Arabidopsis thaliana (L.) Heynh. AtCNGC10 bound CaM in a C-terminal subregion that contains a basic amphiphillic structure characteristic of CaM-binding proteins and that also overlaps with the predicted CN-binding domain. AtCNGC10 is insensitive to the broad-range K+ channel blocker, tetraethylammonium, and lacks a typical K+-signature motif. However, AtCNGC10 complemented K+ channel uptake mutants of Escherichia coli (LB650), yeast (Saccharomyces cerevisiae CY162) and Arabidopsis (akt1-1). Sense 35S-AtCNGC10 transformed into the Arabidopsis akt1-1 mutant, grew 1.7-fold better on K+-limited medium relative to the vector control. Coexpression of CaM and AtCNGC10 in E. coli showed that Ca2+ / CaM inhibited cell growth by 40%, while cGMP reversed the inhibition by Ca2+ / CaM, in a AtCNGC10-dependent manner. AtCNGC10 did not confer tolerance to Cs+ in E. coli, however, it confers tolerance to toxic levels of Na+ and Cs+ in the yeast K+ uptake mutant grown on low K+ medium. Antisense AtCNGC10 plants had 50% less potassium than wild type Columbia. Taken together, the studies from three evolutionarily diverse species demonstrated a role for the CaM-binding channel, AtCNGC10, in mediating the uptake of K+ in plants.
RESUMEN
A full-length cDNA, PPRG2, representing a gene highly expressed in dodder (Cuscuta trifolii Bab et. Gibs)-infected alfalfa (Medicago sativa L.) stems was isolated by differential screening. The predicted protein contains 157 amino acids and belongs to the PR-10 family of the pathogenesis-related genes with putative ribonuclease activities. Northern hybridizations showed that PPRG2 is transcribed in root and crops of uninfected alfalfa and is induced not only by dodder attack but also by bacterial infections and a large variety of environmental stresses.