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1.
EMBO J ; 30(21): 4500-14, 2011 Aug 26.
Artículo en Inglés | MEDLINE | ID: mdl-21873975

RESUMEN

Tumour metastasis suppressors are inhibitors of metastasis but their mechanisms of action are generally not understood. We previously showed that the suppressor Raf kinase inhibitory protein (RKIP) inhibits breast tumour metastasis in part via let-7. Here, we demonstrate an integrated approach combining statistical analysis of breast tumour gene expression data and experimental validation to extend the signalling pathway for RKIP. We show that RKIP inhibits let-7 targets (HMGA2, BACH1) that in turn upregulate bone metastasis genes (MMP1, OPN, CXCR4). Our results reveal BACH1 as a novel let-7-regulated transcription factor that induces matrix metalloproteinase1 (MMP1) expression and promotes metastasis. An RKIP pathway metastasis signature (designated RPMS) derived from the complete signalling cascade predicts high metastatic risk better than the individual genes. These results highlight a powerful approach for identifying signalling pathways downstream of a key metastasis suppressor and indicate that analysis of genes in the context of their signalling environment is critical for understanding their predictive and therapeutic potential.


Asunto(s)
Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/genética , Carcinoma/diagnóstico , Carcinoma/genética , MicroARNs/fisiología , Proteínas de Unión a Fosfatidiletanolamina/fisiología , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/fisiología , Neoplasias de la Mama/patología , Carcinoma/patología , Línea Celular Tumoral , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Análisis por Micromatrices , Modelos Biológicos , Metástasis de la Neoplasia , Proteínas de Unión a Fosfatidiletanolamina/genética , Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Pronóstico , Transducción de Señal/genética , Transducción de Señal/fisiología
2.
Nature ; 462(7272): 522-6, 2009 Nov 26.
Artículo en Inglés | MEDLINE | ID: mdl-19940929

RESUMEN

Translational fidelity, essential for protein and cell function, requires accurate transfer RNA (tRNA) aminoacylation. Purified aminoacyl-tRNA synthetases exhibit a fidelity of one error per 10,000 to 100,000 couplings. The accuracy of tRNA aminoacylation in vivo is uncertain, however, and might be considerably lower. Here we show that in mammalian cells, approximately 1% of methionine (Met) residues used in protein synthesis are aminoacylated to non-methionyl-tRNAs. Remarkably, Met-misacylation increases up to tenfold upon exposing cells to live or non-infectious viruses, toll-like receptor ligands or chemically induced oxidative stress. Met is misacylated to specific non-methionyl-tRNA families, and these Met-misacylated tRNAs are used in translation. Met-misacylation is blocked by an inhibitor of cellular oxidases, implicating reactive oxygen species (ROS) as the misacylation trigger. Among six amino acids tested, tRNA misacylation occurs exclusively with Met. As Met residues are known to protect proteins against ROS-mediated damage, we propose that Met-misacylation functions adaptively to increase Met incorporation into proteins to protect cells against oxidative stress. In demonstrating an unexpected conditional aspect of decoding mRNA, our findings illustrate the importance of considering alternative iterations of the genetic code.


Asunto(s)
Inmunidad Innata , Metionina/metabolismo , Estrés Oxidativo/fisiología , Aminoacilación de ARN de Transferencia/fisiología , Adenoviridae/fisiología , Animales , Código Genético , Células HeLa , Humanos , Ligandos , Metionina/genética , Ratones , Modelos Genéticos , NADPH Oxidasas/metabolismo , Orthomyxoviridae/fisiología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , ARN de Transferencia de Metionina/genética , ARN de Transferencia de Metionina/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Especificidad por Sustrato , Receptores Toll-Like/inmunología , Receptores Toll-Like/metabolismo , Aminoacilación de ARN de Transferencia/efectos de los fármacos
3.
EMBO J ; 28(4): 347-58, 2009 Feb 18.
Artículo en Inglés | MEDLINE | ID: mdl-19153603

RESUMEN

Raf kinase inhibitory protein (RKIP) negatively regulates the MAP kinase (MAPK), G protein-coupled receptor kinase-2, and NF-kappaB signalling cascades. RKIP has been implicated as a metastasis suppressor for prostate cancer, but the mechanism is not known. Here, we show that RKIP inhibits invasion by metastatic breast cancer cells and represses breast tumour cell intravasation and bone metastasis in an orthotopic murine model. The mechanism involves inhibition of MAPK, leading to decreased transcription of LIN28 by Myc. Suppression of LIN28 enables enhanced let-7 processing in breast cancer cells. Elevated let-7 expression inhibits HMGA2, a chromatin remodelling protein that activates pro-invasive and pro-metastatic genes, including Snail. LIN28 depletion and let-7 expression suppress bone metastasis, and LIN28 restores bone metastasis in mice bearing RKIP-expressing breast tumour cells. These results indicate that RKIP suppresses invasion and metastasis in part through a signalling cascade involving MAPK, Myc, LIN28, let-7, and downstream let-7 targets. RKIP regulation of two pluripotent stem cell genes, Myc and LIN28, highlights the importance of RKIP as a key metastasis suppressor and potential therapeutic agent.


Asunto(s)
MicroARNs/metabolismo , Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Línea Celular Tumoral , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , FN-kappa B/metabolismo , Metástasis de la Neoplasia , Trasplante de Neoplasias , Neoplasias de la Próstata/metabolismo , Transducción de Señal
4.
Methods Mol Biol ; 661: 497-505, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20812004

RESUMEN

Maintaining the integrity of the cell cycle is critical for ensuring that cells only undergo DNA replication and proliferation under controlled conditions in response to discrete stimuli. One mechanism by which the fidelity of this process is guaranteed is through the activation of cell cycle checkpoints. The mitotic spindle checkpoint, which is regulated by Aurora B kinase, ensures proper kinetochore attachment to chromosomes leading to equal distribution of chromosomes to daughter cells. We demonstrated that the mitogen-activated protein kinase (MAPK) cascade regulates mitotic progression and the spindle checkpoint. As demonstrated by immunofluorescence at kinetochores, depletion of Raf Kinase Inhibitory Protein (RKIP), an inhibitor of Raf/MEK/ERK signaling, causes an increase in MAPK activity that inhibits Aurora B kinase activity. By monitoring mitotic index and transit time from nuclear envelope breakdown to anaphase, we demonstrated that RKIP depletion leads to a defective spindle checkpoint and genomic instability, particularly in response to drugs that disrupt microtubule function.


Asunto(s)
Técnica del Anticuerpo Fluorescente/métodos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mitosis , Células HeLa , Humanos , Immunoblotting , Coloración y Etiquetado , Timidina/metabolismo
5.
PLoS One ; 4(6): e6028, 2009 Jun 24.
Artículo en Inglés | MEDLINE | ID: mdl-19551145

RESUMEN

BACKGROUND: Raf Kinase Inhibitory Protein (RKIP, also PEBP1), a member of the Phosphatidylethanolamine Binding Protein family, negatively regulates growth factor signaling by the Raf/MAP kinase pathway. Since an organic compound, locostatin, was reported to bind RKIP and inhibit cell migration by a Raf-dependent mechanism, we addressed the role of RKIP in locostatin function. METHODS/FINDINGS: We analyzed locostatin interaction with RKIP and examined the biological consequences of locostatin binding on RKIP function. NMR studies show that a locostatin precursor binds to the conserved phosphatidylethanolamine binding pocket of RKIP. However, drug binding to the pocket does not prevent RKIP association with its inhibitory target, Raf-1, nor affect RKIP phosphorylation by Protein Kinase C at a regulatory site. Similarly, exposure of wild type, RKIP-depleted HeLa cells or RKIP-deficient (RKIP(-/-)) mouse embryonic fibroblasts (MEFs) to locostatin has no effect on MAP kinase activation. Locostatin treatment of wild type MEFs causes inhibition of cell migration following wounding. RKIP deficiency impairs migration further, indicating that RKIP protects cells against locostatin-mediated inhibition of migration. Locostatin treatment of depleted or RKIP(-/-) MEFs reveals cytoskeletal disruption and microtubule abnormalities in the spindle. CONCLUSIONS/SIGNIFICANCE: These results suggest that locostatin's effects on cytoskeletal structure and migration are caused through mechanisms independent of its binding to RKIP and Raf/MAP kinase signaling. The protective effect of RKIP against drug inhibition of migration suggests a new role for RKIP in potentially sequestering toxic compounds that may have deleterious effects on cells.


Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Oxazolidinonas/farmacología , Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Células HeLa , Humanos , Ratones , Modelos Biológicos , Unión Proteica , Proteínas Proto-Oncogénicas c-raf/metabolismo , Ratas , Transducción de Señal
6.
Mol Cell ; 23(4): 561-74, 2006 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16916643

RESUMEN

Raf kinase inhibitory protein (RKIP or PEBP) is an inhibitor of the Raf/MEK/MAP kinase signaling cascade and a suppressor of cancer metastasis. We now show that RKIP associates with centrosomes and kinetochores and regulates the spindle checkpoint in mammalian cells. RKIP depletion causes decreases in the mitotic index, the number of metaphase cells, and traversal times from nuclear envelope breakdown to anaphase, and an override of mitotic checkpoints induced by spindle poisons. Raf-1 depletion or MEK inhibition reverses the reduction in the mitotic index, whereas hyperactivation of Raf mimics the RKIP-depletion phenotype. Finally, RKIP depletion or Raf hyperactivation reduces kinetochore localization and kinase activity of Aurora B, a regulator of the spindle checkpoint. These results indicate that RKIP regulates Aurora B kinase and the spindle checkpoint via the Raf-1/MEK/ERK cascade and demonstrate that small changes in the MAP kinase (MAPK) pathway can profoundly impact the fidelity of the cell cycle.


Asunto(s)
Proteína de Unión a Andrógenos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Huso Acromático/metabolismo , Proteína de Unión a Andrógenos/deficiencia , Animales , Aurora Quinasa B , Aurora Quinasas , Centrosoma/metabolismo , Cromatina/efectos de los fármacos , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células HeLa , Humanos , Cinetocoros/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Metafase/efectos de los fármacos , Nocodazol/farmacología , Paclitaxel/farmacología , Proteínas de Unión a Fosfatidiletanolamina , Fosforilación , Prostateína , Transporte de Proteínas , Ratas , Secretoglobinas , Huso Acromático/efectos de los fármacos , Células Tumorales Cultivadas , Uteroglobina , Quinasas raf/metabolismo
7.
J Biol Chem ; 279(42): 43392-402, 2004 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-15297455

RESUMEN

Store-operated calcium entry (SOCE) and TRPC protein expression were investigated in the rat-derived hippocampal H19-7 cell line. Thapsigargin-stimulated Ba2+ entry and the expression of TRPC1, TRPC3, TRPC4, TRPC5, TRPC6, and TRPC7 mRNA and protein were observed in proliferating H19-7 cells. When cells were placed under differentiating conditions, a change in TRPC homolog expression profile occurred. The expression of TRPC1 and TRPC3 mRNA and protein dramatically increased, while the expression of TRPC4 and TRPC7 mRNA and protein dramatically decreased; in parallel a 3.4-fold increase in the level of thapsigargin-stimulated Ba2+ entry was observed and found to be inhibited by 2-aminoethoxydiphenylborane. The selective suppression of TRPC protein levels by small interfering RNA (siRNA) approaches indicated that TRPC1 and TRPC3 are involved in mediating SOCE in proliferating H19-7 cells. Although TRPC4 and TRPC7 are expressed at much higher levels than TRPC1 and TRPC3 in proliferating cells, they do not appear to mediate SOCE. The co-expression of siRNA specific for TRPC1 and TRPC3 in proliferating cells inhibited approximately the same amount of SOCE as observed with expression of either siRNA alone, suggesting that TRPC1 and TRPC3 work in tandem to mediate SOCE. Under differentiating conditions, co-expression of siRNA for TRPC1 and TRPC3 blocked the normal 3.4-fold increase in SOCE and in turn blocked the differentiation of H19-7 cells. This study suggests that placing H19-7 cells under differentiating conditions significantly alters TRPC gene expression and increases the level of SOCE and that this increase in SOCE is necessary for cell differentiation.


Asunto(s)
Canales de Calcio/fisiología , Señalización del Calcio/fisiología , Calcio/metabolismo , Proteínas de Transporte de Catión/fisiología , Diferenciación Celular/fisiología , Hipocampo/fisiología , Canales Iónicos/fisiología , Neuronas/citología , Animales , Secuencia de Bases , Transporte Biológico , Canales de Calcio/genética , Proteínas de Transporte de Catión/genética , División Celular/fisiología , Línea Celular , Cartilla de ADN , Hipocampo/citología , Canales Iónicos/genética , Cinética , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Ratas , Canales Catiónicos TRPC , Tapsigargina/farmacología
8.
J Biol Chem ; 278(15): 13061-8, 2003 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-12551925

RESUMEN

Protein kinase C (PKC) regulates activation of the Raf-1 signaling cascade by growth factors, but the mechanism by which this occurs has not been elucidated. Here we report that one mechanism involves dissociation of Raf kinase inhibitory protein (RKIP) from Raf-1. Classic and atypical but not novel PKC isoforms phosphorylate RKIP at serine 153 (Ser-153). RKIP Ser-153 phosphorylation by PKC either in vitro or in response to 12-O-tetradecanoylphorbol-13-acetate or epidermal growth factor causes release of RKIP from Raf-1, whereas mutant RKIP (S153V or S153E) remains bound. Increased expression of PKC can rescue inhibition of the mitogen-activated protein (MAP) kinase signaling cascade by wild-type but not mutant S153V RKIP. Taken together, these results constitute the first model showing how phosphorylation by PKC relieves a key inhibitor of the Raf/MAP kinase signaling cascade and may represent a general mechanism for the regulation of MAP kinase pathways.


Asunto(s)
Proteína de Unión a Andrógenos , Proteínas Portadoras/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas c-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-raf/metabolismo , Animales , Secuencia de Bases , Proteínas Portadoras/química , Proteínas Portadoras/genética , Bovinos , Línea Celular Transformada , Cristalografía por Rayos X , Cartilla de ADN , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Humanos , Proteínas de Unión a Fosfatidiletanolamina , Proteínas de Transferencia de Fosfolípidos , Fosforilación , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Transfección
9.
Am J Respir Crit Care Med ; 169(6): 703-11, 2004 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-14693670

RESUMEN

We developed clonal cell lines of human bronchial smooth muscle origin by retroviral transduction of temperature-sensitive simian virus 40 large tumor (T) antigen. These cells show increased growth potential at 33 degrees C, but on shift to the nonpermissive temperature (39 degrees C), they show diminished or arrested growth. In addition to the expected reduction in the level of large T antigen, cells shifted to 39 degrees C show increased expression of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1), characteristic of cells arrested in G1 of the cell cycle. Shifted cells undergo a process of cell hypertrophy, as demonstrated by increased time of flight and forward scatter, as well as increased expression of the contractile proteins alpha-smooth muscle actin, myosin light chain kinase, and SM22. Changes in contractile protein expression were regulated primarily in a posttranscriptional manner. Phosphatidylinositol 3-kinase activity was increased in shifted cells, and chemical inhibition of phosphatidylinositol 3-kinase attenuated alpha-actin and myosin light-chain kinase expression. We have developed clonal cell lines of human bronchial smooth muscle origin that may be useful for the study of airway smooth muscle biology. Furthermore, we demonstrate that arrest of airway smooth muscle cell cycle traversal can induce cellular hypertrophy, which parallels changes observed in the airways of patients with severe asthma.


Asunto(s)
Bronquios/citología , Bronquios/fisiología , Línea Celular Transformada/fisiología , Células Clonales/fisiología , Miocitos del Músculo Liso/fisiología , Antígenos Virales de Tumores , Asma/genética , Técnicas de Cultivo de Célula , División Celular/fisiología , Transformación Celular Viral , Humanos , Hipertrofia/genética , Oncogenes , Fenotipo , Virus 40 de los Simios/inmunología , Temperatura
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