RESUMEN

During spermatogenesis, the blood-testis barrier (BTB) undergoes cyclic remodeling that is crucial to support the transport of preleptotene spermatocytes across the immunological barrier at stage VIII to IX of the epithelial cycle. Studies have shown that this timely remodeling of the BTB is supported by several endogenously produced barrier modifiers across the seminiferous epithelium, which include the F5-peptide and the ribosomal protein S6 [rpS6; a downstream signaling molecule of the mammalian target of rapamycin complex 1 (mTORC1)] signaling protein. Herein, F5-peptide and a quadruple phosphomimetic (and constitutively active) mutant of rpS6 [i.e., phosphorylated (p-)rpS6-MT] that are capable of inducing reversible immunological barrier remodeling, by making the barrier "leaky" transiently, were used for their overexpression in the testis to induce BTB opening. We sought to examine whether this facilitated the crossing of the nonhormonal male contraceptive adjudin at the BTB when administered by oral gavage, thereby effectively improving its BTB transport to induce germ cell adhesion and aspermatogenesis. Indeed, it was shown that combined overexpression of F5-peptide and p-rpS6-MT and a low dose of adjudin, which by itself had no noticeable effects on spermatogenesis, was capable of perturbing the organization of actin- and microtubule (MT)-based cytoskeletons through changes in the spatial expression of actin- and MT-binding/regulatory proteins to the corresponding cytoskeleton. These findings thus illustrate the possibility of delivering drugs to any target organ behind a blood-tissue barrier by modifying the tight junction permeability barrier using endogenously produced barrier modifiers based on findings from this adjudin animal model.

Asunto(s)

Barrera Hematotesticular/metabolismo , Laminina/fisiología , Diana Mecanicista del Complejo 1 de la Rapamicina/fisiología , Proteína S6 Ribosómica/fisiología , Actinas , Animales , Transporte Biológico/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Hidrazinas/farmacología , Indazoles/farmacología , Masculino , Fragmentos de Péptidos/fisiología , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Espermatogénesis/efectos de los fármacos , Testículo/efectos de los fármacos , Proteínas de Uniones Estrechas/análisis

RESUMEN

Some amino acids (AA) act through several signalling pathways and mechanisms to mediate the control of gene expression at the translation level, and the regulation occurs, specifically, on the initiation and the signalling pathways for translation. The translation of mRNA to protein synthesis proceeds through the steps of initiation and elongation, and AA act as important feed-forward activators that are involved in many pathways, such as the sensing and the transportation of AA by cells, in these steps in many tissues of mammals. For the translation, phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) is a critical molecule that controls the translation initiation and its functions can be regulated by some AA. Another control point in the mRNA binding step in the translation initiation is at the regulation by mammalian target of rapamycin, which requires a change of phosphorylation status of ribosomal protein S6. In fact, the change of phosphorylation status of ribosomal protein S6 might be involved in global protein synthesis. The present review summarises recent work on the molecular mechanisms of the regulation of protein synthesis by AA and highlights new findings.

Asunto(s)

Aminoácidos/fisiología , Regulación de la Expresión Génica/fisiología , Biosíntesis de Proteínas/genética , Animales , Arginina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Leucina/farmacología , Extensión de la Cadena Peptídica de Translación/fisiología , Fosforilación/fisiología , ARN Mensajero/genética , Proteína S6 Ribosómica/fisiología , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/química , Serina-Treonina Quinasas TOR/fisiología , eIF-2 Quinasa/fisiología

RESUMEN

The blood-testis barrier (BTB), conferred by Sertoli cells in the mammalian testis, is an important ultrastructure that supports spermatogenesis. Studies using animal models have shown that a disruption of the BTB leads to meiotic arrest, causing defects in spermatogenesis and male infertility. To better understand the regulation of BTB dynamics, we report findings herein to understand the role of ribosomal protein S6 (rpS6), a downstream signaling protein of mammalian target of rapamycin complex 1 (mTORC1), in promoting BTB disruption in the testis in vivo, making the barrier "leaky." Overexpression of wild-type rpS6 (rpS6-WT, the full-length cDNA cloned into the mammalian expression vector pCI-neo) and a constitutively active quadruple phosphomimetic mutant cloned into pCI-neo (p-rpS6-MT) vs. control (empty pCI-neo vector) was achieved by transfecting adult rat testes with the corresponding plasmid DNA using a Polyplus in vivo-jetPEI transfection reagent. On the basis of an in vivo functional BTB integrity assay, p-rpS6-MT was found to induce BTB disruption better than rpS6-WT did (and no effects in empty vector control), leading to defects in spermatogenesis, including loss of spermatid polarity and failure in the transport of cells (e.g., spermatids) and organelles (e.g., phagosomes), to be followed by germ exfoliation. More important, rpS6-WT and p-rpS6-MT exert their disruptive effects through changes in the organization of actin- and microtubule (MT)-based cytoskeletons, which are mediated by changes in the spatiotemporal expression of actin- and MT-based binding and regulatory proteins. In short, mTORC1/rpS6 signaling complex is a regulator of spermatogenesis and BTB by modulating the organization of the actin- and MT-based cytoskeletons.

Asunto(s)

Barrera Hematotesticular/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/fisiología , Proteína S6 Ribosómica/fisiología , Espermatogénesis/fisiología , Testículo/metabolismo , Animales , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Permeabilidad , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Proteína S6 Ribosómica/genética , Transducción de Señal/genética , Espermatogénesis/genética , Testículo/fisiología , Regulación hacia Arriba/genética

RESUMEN

Skeletal muscle mass is an important trait in the animal industry. We previously reported an age-dependent downregulation of the insulin-like growth factor 1 (IGF-1)/Akt/S6 pathway, major protein synthesis pathway, in chicken breast muscle after 1 week of age, despite a continuous increase of breast muscle weight. Myosin heavy chain (HC), a major protein in muscle fiber, has several isoforms depending on chicken skeletal muscle types. HC I (fast-twitch glycolytic type) is known to be expressed in adult chicken breast muscle. However, little is known about the changes in the expression levels of protein synthesis-related factors and HC isoforms in perihatching chicken muscle. In the present study, protein synthesis-related factors, such as IGF-1 messenger RNA (mRNA) levels, phosphorylation of Akt, and phosphorylated S6 content, increased in an age-dependent manner after post-hatch day (D) 0. The mRNA levels of HC I, III and V (fast-twitch glycolytic type) dramatically increased after D0. The increase ratio of breast muscle weight was approximately 1100% from D0 to D7. To our knowledge, these findings provide the first evidence that upregulation of protein synthesis pathway and transcription of fast twitch glycolytic HC isoforms play critical roles in the increase of chicken breast muscle weight during the first week after hatching.

Asunto(s)

Animales Recién Nacidos/genética , Animales Recién Nacidos/metabolismo , Pollos/genética , Pollos/metabolismo , Expresión Génica , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/fisiología , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Biosíntesis de Proteínas/genética , Biosíntesis de Proteínas/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-akt/fisiología , Proteína S6 Ribosómica/metabolismo , Proteína S6 Ribosómica/fisiología , Transducción de Señal/fisiología , Regulación hacia Arriba , Envejecimiento/genética , Envejecimiento/metabolismo , Animales , Femenino , Masculino , Músculo Esquelético/crecimiento & desarrollo , Tamaño de los Órganos/genética , Tamaño de los Órganos/fisiología , Fosforilación , Isoformas de Proteínas/metabolismo

RESUMEN

Retrieval of consolidated memories induces a labile phase during which memory can be disrupted or updated through a reconsolidation process. A central component of behavioral updating during reconsolidation using a retrieval-extinction manipulation (Ret+Ext) is the synaptic removal of a calcium-permeable-α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (CP-AMPARs) in the lateral amygdala-a metabotropic GluR1 receptor (mGluR1) dependent mechanism. In the present study, we investigate the effect of Ret+Ext on the expression of molecular markers that could play a role in the reconsolidation process. Specifically, we tested the effects of Ret+Ext on the global expression of zinc-finger 268 protein (Zif268), a marker previously found to be implicated in memory reconsolidation, to confirm its occurrence after retrieval (Ret) and Ret+Ext. We also evaluated the global expression of phosphorylated ribosomal protein S6 (rpS6P), here proposed as a marker of the mGluR1-mediated memory process induced by Ret+Ext. The expression of both markers (zif268, rpS6P) was assessed by immunolocalization in prelimbic cortex (PRL), infralimbic cortex (IL), ventral subdivision of the lateral amygdala (LA) and hippocampus CA1 (CA1) in fear-conditioned rats. Our results showed that retrieval and Ret+Ext, but not extinction alone, increased Zif268 expression in prefrontal cortex and lateral amygdala. Ret+Ext, but not retrieval, retrieval followed by context exposure or extinction alone, increased the expression of rpS6P in prefrontal cortex and LA. In summary, (i) Zif268 increased after retrieval confirming that reconsolidation is engaged in our conditions, (ii) Zif268 increased after Ret+Ext confirming that it does not simply reflect an extinction or reconsolidation disruption (Zif268 level of expression should be lower in both cases) and (iii) rpS6P increased after Ret+Ext, but not after extinction, suggesting, as expected, a potential mGluR1 mediated molecular mechanism specific for Ret+Ext. Together with the Zif268 increase, our results suggest that the Ret+Ext induced memory process is more similar to reconsolidation updating than extinction facilitation.

Asunto(s)

Complejo Nuclear Basolateral/fisiología , Proteína 1 de la Respuesta de Crecimiento Precoz/fisiología , Extinción Psicológica/fisiología , Miedo/fisiología , Recuerdo Mental/fisiología , Corteza Prefrontal/fisiología , Proteína S6 Ribosómica/fisiología , Estimulación Acústica , Animales , Complejo Nuclear Basolateral/química , Condicionamiento Clásico/fisiología , Proteína 1 de la Respuesta de Crecimiento Precoz/análisis , Proteína 1 de la Respuesta de Crecimiento Precoz/biosíntesis , Masculino , Fosforilación , Corteza Prefrontal/química , Ratas Sprague-Dawley , Proteína S6 Ribosómica/análisis , Proteína S6 Ribosómica/biosíntesis

Asunto(s)

Complejos Multiproteicos/fisiología , Isquemia Miocárdica/prevención & control , Proteínas Proto-Oncogénicas c-akt/fisiología , Proteína S6 Ribosómica/fisiología , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/fisiología , Animales , Masculino , Diana Mecanicista del Complejo 2 de la Rapamicina

RESUMEN

RATIONALE: There is tight coupling between Akt activation and suppression of cell death. Full Akt activation requires mammalian target of rapamycin complex 2 (mTORC2), but the regulation of mTORC2 is unclear. OBJECTIVE: To gain new insights into mechanisms of mTORC2/Akt signaling. METHODS AND RESULTS: The role of mTORC2 in cardioprotection was examined. In perfused mouse hearts, ischemic preconditioning increased mTORC2 activity, leading to phosphorylation of Akt on Ser473. The protective effect of ischemic preconditioning was lost by pretreatment with dual mTORC inhibitors but not with rapamycin, an mTORC1 inhibitor, which indicates the fundamental role of mTORC2 activation in cardioprotection. Next, the regulation and downstream targets of mTORC2/Akt signaling were explored. We have found that ischemic preconditioning and other Akt activators (insulin and opioids) result in phosphorylation of ribosomal protein S6 (Rps6) at Ser235/236 in mouse hearts and neonatal rat ventricular myocytes. Rps6 interacts with components of mTORC2, and siRNA-mediated knockdown of Rps6 attenuates insulin-induced mTORC2 activation and Akt-Ser473 phosphorylation. On the other hand, Rps6 overexpression enhanced Akt-Ser473 phosphorylation, indicating that Rps6 activation amplifies mTORC2/Akt signaling. Disruption of the Rps6/mTORC2 pathway by knockdown of Rps6 or rictor abrogated insulin-induced cytoprotection against oxidative stress. Although rapamycin blocks Rps6-dependent mTORC2 activation, mTORC2 is still activated by an alternative signaling pathway, demonstrating the redundancy in cardioprotective signaling. CONCLUSIONS: Activation of mTORC2 plays a pivotal role in cardioprotection, and Rps6 is a convergence point of cardioprotective signaling, providing positive feedback regulation of mTORC2/Akt signaling.

Asunto(s)

Complejos Multiproteicos/fisiología , Isquemia Miocárdica/prevención & control , Proteínas Proto-Oncogénicas c-akt/fisiología , Proteína S6 Ribosómica/fisiología , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/fisiología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Retroalimentación Fisiológica/fisiología , Precondicionamiento Isquémico Miocárdico , Masculino , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Ratones Endogámicos C57BL , Isquemia Miocárdica/fisiopatología , Fosforilación/fisiología

RESUMEN

PURPOSE: To assess the functional consequences of silencing of tuberin, an inhibitor of the mTOR signaling pathway, in a preclinical model of retinitis pigmentosa (RP) in order to test the hypothesis that insufficient induction of the protein kinase B (PKB)-regulated tuberin/mTOR self-survival pathway initiates apoptosis. METHODS: In an unbiased genome-scale approach, kinase peptide substrate arrays were used to analyze self-survival pathways at the onset of photoreceptor degeneration. The mutant Pde6b(H620Q)/Pde6b(H620Q) at P14 and P18 photoreceptor outer segment (OS) lysates were labeled with P-ATP and hybridized to an array of 1,164 different synthetic peptide substrates. At this stage, OS of Pde6b(H620Q)/Pde6b(H620Q) rods are morphologically normal. In vitro kinase assays and immunohistochemistry were used to validate phosphorylation. Short hairpin RNA (shRNA) gene silencing was used to validate tuberin's role in regulating survival. RESULTS: At the onset of degeneration, 162 peptides were differentially phosphorylated. Protein kinases A, G, C (AGC kinases), and B exhibited increased activity in both peptide array and in vitro kinase assays. Immunohistochemical data confirmed altered phosphorylation patterns for phosphoinositide-dependent kinase-1 (PDK1), ribosomal protein S6 (RPS6), and tuberin. Tuberin gene silencing rescued photoreceptors from degeneration. CONCLUSIONS: Phosphorylation of tuberin and RPS6 is due to the upregulated activity of PKB. PKB/tuberin cell growth/survival signaling is activated before the onset of degeneration. Substrates of the AGC kinases in the PKB/tuberin pathway are phosphorylated to promote cell survival. Knockdown of tuberin, the inhibitor of the mTOR pathway, increased photoreceptor survival and function in a preclinical model of RP.

Asunto(s)

Retinitis Pigmentosa/fisiopatología , Proteínas Supresoras de Tumor/fisiología , Animales , Apoptosis/fisiología , Modelos Animales de Enfermedad , Silenciador del Gen , Ratones , Ratones Mutantes , Fosforilación , Fosfotransferasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína S6 Ribosómica/fisiología , Transducción de Señal/fisiología , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genética

RESUMEN

The molecular mechanism(s) linking tumorigenesis and morphological alterations in the nucleolus are presently coming into focus. The nucleolus is the cellular organelle in which the formation of ribosomal subunits occurs. Ribosomal biogenesis occurs through the transcription of ribosomal RNA (rRNA), rRNA processing and production of ribosomal proteins. An error in any of these processes may lead to deregulated cellular translation, evident in multiple cancers and 'ribosomopathies'. Deregulated protein synthesis may be achieved through the overexpression of ribosomal proteins as seen in primary leukemic blasts with elevated levels of ribosomal proteins S11 and S14. In this study, we demonstrate that ribosomal protein S6 (RPS6) is highly expressed in primary diffuse large B-cell lymphoma (DLBCL) samples. Genetic modulation of RPS6 protein levels with specifically targeted short hairpin RNA (shRNA) lentiviruses led to a decrease in the actively proliferating population of cells compared with control shRNA. Low-dose rapamycin treatments have been shown to affect the translation of 5' terminal oligopyrimidine (5' TOP) tract mRNA, which encodes the translational machinery, implicating RPS6 in 5' TOP translation. Recently, it was shown that disruption of 40S ribosomal biogenesis through specific small inhibitory RNA knockdown of RPS6 defined RPS6 as a critical regulator of 5' TOP translation. For the first time, we show that RPS6 associates with multiple mRNAs containing a 5' TOP tract. These findings expand our understanding of the mechanism(s) involved in ribosomal biogenesis and deregulated protein synthesis in DLBCL.

Asunto(s)

Linfoma de Células B Grandes Difuso/metabolismo , Secuencia de Oligopirimidina en la Región 5' Terminal del ARN/genética , Proteína S6 Ribosómica/fisiología , Línea Celular Tumoral , Nucléolo Celular/fisiología , Endorribonucleasas/análisis , Humanos , Fenotipo , Proteínas de Unión a Poli(A)/análisis , Biosíntesis de Proteínas , ARN Mensajero/genética , Proteína S6 Ribosómica/análisis , Ribosomas/fisiología , Sirolimus/farmacología , Antígeno Intracelular 1 de las Células T

RESUMEN

Deregulated nutrient signaling plays pivotal roles in body ageing and in diabetic complications; biochemical cascades linking energy dysmetabolism to cell damage and loss are still incompletely clarified, and novel molecular paradigms and pharmacological targets critically needed. We provide evidence that in the retrovirus-packaging cell line HEK293-T Phoenix, massive cell death in serum-free medium is remarkably prevented or attenuated by either glucose or aminoacid withdrawal, and by the glycolysis inhibitor 2-deoxy-glucose. A similar protection was also elicited by interference with mitochondrial function, clearly suggesting involvement of energy metabolism in increased cell survival. Oxidative stress did not account for nutrient toxicity on serum-starved cells. Instead, nutrient restriction was associated with reduced activity of the mTOR/S6 Kinase cascade. Moreover, pharmacological and genetic manipulation of the mTOR pathway modulated in an opposite fashion signaling to S6K/S6 and cell viability in nutrient-repleted medium. Additionally, stimulation of the AMP-activated Protein Kinase concomitantly inhibited mTOR signaling and cell death, while neither event was affected by overexpression of the NAD+ dependent deacetylase Sirt-1, another cellular sensor of nutrient scarcity. Finally, blockade of the mTOR cascade reduced hyperglycemic damage also in a more pathophysiologically relevant model, i.e. in human umbilical vein endothelial cells (HUVEC) exposed to hyperglycemia. Taken together these findings point to a key role of the mTOR/S6K cascade in cell damage by excess nutrients and scarcity of growth-factors, a condition shared by diabetes and other ageing-related pathologies.

Asunto(s)

Supervivencia Celular/fisiología , Privación de Alimentos/fisiología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Quinasas Activadas por AMP/fisiología , Antimetabolitos/administración & dosificación , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Medio de Cultivo Libre de Suero , Desoxiglucosa/administración & dosificación , Células HEK293 , Humanos , Mitocondrias/fisiología , Estrés Oxidativo/fisiología , Proteína S6 Ribosómica/fisiología , Proteínas Quinasas S6 Ribosómicas/fisiología , Transducción de Señal/fisiología , Sirtuina 1/fisiología , Serina-Treonina Quinasas TOR/toxicidad

RESUMEN

We have previously shown that in Dictyostelium cells a 32 kDa protein is rapidly and completely dephosphorylated in response to starvation that is essential for the initiation of differentiation (Akiyama & Maeda 1992). In the present work, this phosphoprotein was identified as a homologue (Dd-RPS6) of ribosomal protein S6 (RPS6) that is an essential member for protein synthesis. As expected, Dd-RPS6 seems to be absolutely required for cell survival, because we failed to obtain antisense-RNA mediated cells as well as Dd-rps6-null cells by homologous recombination in spite of many trials. In many kinds of cell lines, RPS6 is known to be located in the nucleus and cytosol, but Dd-RPS6 is predominantly located in the cell cortex with cytoskeletons, and in the contractile ring of just-dividing cells. In this connection, the overexpression of Dd-RPS6 greatly impairs cytokinesis during axenic shake-cultures in growth medium, resulting in the formation of multinucleate cells. Much severe impairment of cytokinesis was observed when Dd-RPS6-overexpressing cells (Dd-RPS6(OE) cells) were incubated on a living Escherichia coli lawn. The initiation of differentiation triggered by starvation was also delayed in Dd-RPS6(OE) cells. In addition, Dd-RPS6(OE) cells exhibit defective differentiation into prespore cells and spores during late development. Thus, it is likely that the proper expression of Dd-RPS6 may be of importance for the normal progression of late differentiation as well as for the initiation of differentiation.

Asunto(s)

Dictyostelium/fisiología , Proteínas Protozoarias/fisiología , Proteína S6 Ribosómica/fisiología , Western Blotting , Ciclo Celular , Diferenciación Celular , Proliferación Celular , Citocinesis , Dictyostelium/citología , Dictyostelium/genética , Técnica del Anticuerpo Fluorescente , Expresión Génica , Fosforilación , Proteínas Protozoarias/genética , Proteína S6 Ribosómica/genética , Inanición

RESUMEN

The phosphorylation of ribosomal protein S6 (rpS6), which occurs in response to a wide variety of stimuli on five evolutionarily conserved serine residues, has attracted much attention since its discovery more than three decades ago. However, despite a large body of information on the respective kinases and the signal transduction pathways, the role of this phosphorylation remained obscure. It is only recent that targeting the genes encoding rpS6, the phosphorylatable serine residues or the respective kinases that the unique role of rpS6 and its posttranslational modification have started to be elucidated. This review focuses primarily on the critical role of rpS6 for mouse development, the pathways that transduce various signals into rpS6 phosphorylation, and the physiological functions of this modification. The mechanism(s) underlying the diverse effects of rpS6 phosphorylation on cellular and organismal physiology has yet to be determined. However, a model emerging from the currently available data suggests that rpS6 phosphorylation operates, at least partly, by counteracting positive signals simultaneously induced by rpS6 kinase, and thus might be involved in fine-tuning of the cellular response to these signals.

Asunto(s)

Proteína S6 Ribosómica/fisiología , Secuencia de Aminoácidos , Animales , Diferenciación Celular/fisiología , Tamaño de la Célula , Evolución Molecular , Glucosa/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Fosforilación , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína S6 Ribosómica/química , Proteína S6 Ribosómica/genética , Transducción de Señal , Factores de Transcripción/metabolismo

RESUMEN

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a potent inducer of apoptosis in tumor cells and holds a promise as a therapeutic agent against cancer. To elucidate the death signaling evoked by TRAIL, we performed a functional genetic screening and rescued TRAIL-resistant Jurkat clones harboring ribosomal protein S6 (rpS6) cDNA in anti-sense frame. Reduction of rpS6 expression in Jurkat and HeLa cells attenuated apoptosis induced by TRAIL, but not those by other cell death signals, including tumor necrosis factor-alpha and cycloheximide, etoposide, doxorubicin, tunicamycin and staurosporine. Death receptor (DR) 4, but not DR5, was downregulated in rpS6 knockdown cells. Conversely, the sensitivity to TRAIL was increased by the ectopic expression of wild-type rpS6 and further by phospho-defective rpS6 mutant (S6-SS235,6AA), but not by phospho-mimic rpS6 mutant (S6-SS235,6DD). Also, unphosphorylatable rpS6 knock-in mouse embryo fibroblasts (rpS6(P-/-) MEFs) were more sensitive to TRAIL than control MEFs. In addition, SKHep-1 tumor cells, which express less phospho-rpS6 and are more sensitive to TRAIL than other tumor cells, became effectively desensitized to TRAIL after rpS6 knockdown. These results suggest that rpS6, especially in its unphosphorylated form, is a selective mediator of TRAIL-induced apoptosis.

Asunto(s)

Apoptosis , Proteína S6 Ribosómica/fisiología , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Animales , Antineoplásicos/farmacología , ADN Complementario/metabolismo , Células HeLa , Humanos , Células Jurkat , Ratones , Ratones Transgénicos , Fosforilación , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal

RESUMEN

S6 kinase (S6K) deletion in metazoans causes small cell size, insulin hypersensitivity, and metabolic adaptations; however, the underlying molecular mechanisms are unclear. Here we show that S6K-deficient skeletal muscle cells have increased AMP and inorganic phosphate levels relative to ATP and phosphocreatine, causing AMP-activated protein kinase (AMPK) upregulation. Energy stress and muscle cell atrophy are specifically triggered by the S6K1 deletion, independent of S6K2 activity. Two known AMPK-dependent functions, mitochondrial biogenesis and fatty acid beta-oxidation, are upregulated in S6K-deficient muscle cells, leading to a sharp depletion of lipid content, while glycogen stores are spared. Strikingly, AMPK inhibition in S6K-deficient cells restores cell growth and sensitivity to nutrient signals. These data indicate that S6K1 controls the energy state of the cell and the AMPK-dependent metabolic program, providing a mechanism for cell mass accumulation under high-calorie diet.

Asunto(s)

Adaptación Fisiológica , Adenilato Quinasa/metabolismo , Eliminación de Gen , Músculo Esquelético/fisiología , Proteína S6 Ribosómica/fisiología , Adenilato Quinasa/antagonistas & inhibidores , Adenilato Quinasa/genética , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/metabolismo , Animales , Células Cultivadas , Metabolismo Energético , Activación Enzimática , Ayuno , Prueba de Tolerancia a la Glucosa , Proteínas Fluorescentes Verdes/metabolismo , Hipoglucemiantes/metabolismo , Immunoblotting , Espectroscopía de Resonancia Magnética , Ratones , Ratones Noqueados , Fibras Musculares Esqueléticas/fisiología , Palmitatos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleótidos/metabolismo

RESUMEN

The capacity to detect and appropriately respond to many different stresses that interfere with functional homeostasis is essential for survival. Recent evidence suggests that the nucleolus, the site of ribosome biogenesis, plays a critical role in sensing and responding to both external and internal stresses. To understand these processes, we have recently used a genetically defined in vivo mouse model in which ribosome biogenesis could be manipulated during oogenesis and embryo development. In these mice ribosomal biosynthesis is impaired by a conditional deletion of one allele of the gene encoding 40S ribosomal protein S6. Embryos from these animals fail during gastrulation, apparently due to a p53-dependent checkpoint being triggered, rather than a deficit in translational capacity. These findings imply that molecular mechanisms have evolved during mammalian evolution to strongly guard against potential heterozygosity for ribosomal protein genes.

Asunto(s)

Regulación de la Expresión Génica , Haplotipos/fisiología , Proteína S6 Ribosómica/deficiencia , Proteína S6 Ribosómica/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , Dosificación de Gen/fisiología , Humanos , Ratones , Proteína S6 Ribosómica/fisiología

RESUMEN

In multicellular organisms, growth and metabolism are controlled by extracellular signals, such as insulin and insulin-like growth factors (IGFs). Depending on nutrient availability, these factors regulate cell number, cell size, storage of lipids, proteins and sugars. Here we will review recent literature on the intracellular signal transduction pathways regulating the anabolic responses in skeletal muscles. Emphasis will be put on three serine/threonine kinases, mTOR, Akt and S6 Kinase (S6K), and their role in the integration of environmental cues and the coordination of muscle growth.

Asunto(s)

Tamaño Corporal/genética , Células Musculares/fisiología , Músculo Esquelético/citología , Proteínas Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Proteínas Quinasas S6 Ribosómicas/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Recuento de Células , Eliminación de Gen , Humanos , Ratones , Células Musculares/efectos de los fármacos , Fosfatidilinositol 3-Quinasas , Proteínas/fisiología , Proteínas Proto-Oncogénicas c-akt/deficiencia , Proteínas Proto-Oncogénicas c-akt/genética , Proteína Reguladora Asociada a mTOR , Proteína S6 Ribosómica/fisiología , Proteínas Quinasas S6 Ribosómicas/deficiencia , Proteínas Quinasas S6 Ribosómicas/genética , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Serina-Treonina Quinasas TOR

RESUMEN

Functional ribosomes synthesize proteins in all living cells and are composed of two labile associated subunits, which are made of rRNA and ribosomal proteins. The rRNA of the small 40S subunit (SSU) of the functional eukaryotic 80S ribosome decodes the mRNA molecule and the large 60S subunit (LSU) rRNA catalyzes protein synthesis. Recent fine structure determinations of the ribosome renewed interest in the role of ribosomal proteins in modulation of the core ribosomal functions. RpL10/Grc5p is a component of the LSU and is a multifunctional translational regulator, operating in 60S subunit biogenesis, 60S subunit export and 60S subunit joining with the 40S subunit. Here, we report that rpL10/Grc5p functionally interacts with the nuclear export factor Nmd3p in modulation of the cellular polysome complement and with the small subunit protein rpS6 in subunit joining and differential protein expression.

Asunto(s)

Proteína S6 Ribosómica/fisiología , Proteínas Ribosómicas/fisiología , Ribosomas/fisiología , Levaduras/fisiología , Secuencia de Bases , ADN de Hongos/química , ADN de Hongos/genética , Electroforesis en Gel Bidimensional , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteína Ribosómica L10 , Proteína S6 Ribosómica/genética , Proteínas Ribosómicas/genética , Ribosomas/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Levaduras/genética

RESUMEN

BACKGROUND: At distinct times during embryonic development and after vascular injury, smooth muscle cells (SMCs) exhibit a highly proliferative, serum-independent growth phenotype. The aim of the present study was to evaluate the functional role of S6 ribosomal protein (S6RP) and upstream positive and negative regulators in the control of SMC serum-independent growth. METHODS AND RESULTS: We previously reported increased expression of S6RP mRNA was associated with this unique growth phenotype. Using immunohistochemistry and Western blot analysis, we report high levels of total and phospho-S6RP and increased levels of Akt and p70S6K phosphorylation, upstream positive regulators of S6RP, in rat embryonic aortas and adult balloon-injured carotid arteries compared with quiescent adult aortas and uninjured carotid arteries. Western blot analysis demonstrated that cultured embryonic and neointimal SMCs that exhibited serum-independent growth capabilities expressed high levels of S6RP and constitutively active Akt, mTOR, and p70S6K. Pharmacological and molecular inhibition of phosphatidylinositol 3-kinase (PI3K) signaling pathways, using PI3K inhibitors, rapamycin, or dominant-negative Akt adenovirus, suppressed embryonic and neointimal SMC serum-independent growth. Finally, decreased activity of PTEN, an endogenous negative regulator of PI3K signaling, was associated with high in vivo SMC growth rates, and morpholino-mediated loss of endogenous PTEN induced a serum-independent growth phenotype in cultured serum-dependent SMCs. CONCLUSIONS: The possibility exists that cells that exhibit a distinct embryonic-like growth phenotype different from traditional SMCs are major contributors to intimal thickening. Growth of SMCs that exhibit this phenotype is dependent on constitutive Akt and mTOR/p70S6K signaling and is actively inhibited through the timed acquisition of the endogenously produced growth suppressor PTEN.

Asunto(s)

Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Proteínas Quinasas/fisiología , Proteínas Serina-Treonina Quinasas , Proteínas Tirosina Fosfatasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Quinasas S6 Ribosómicas 70-kDa/fisiología , Proteína S6 Ribosómica/fisiología , Transducción de Señal/fisiología , Túnica Íntima/metabolismo , Androstadienos/farmacología , Animales , Aorta/embriología , Aorta/crecimiento & desarrollo , Traumatismos de las Arterias Carótidas/metabolismo , Cateterismo/efectos adversos , División Celular , Células Cultivadas/metabolismo , Cromonas/farmacología , Medio de Cultivo Libre de Suero/farmacología , Inhibidores Enzimáticos/farmacología , Morfolinas/farmacología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Oligonucleótidos Antisentido/farmacología , Fosfohidrolasa PTEN , Fenotipo , Fosfatidilinositol 3-Quinasas/fisiología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Inhibidores de Proteínas Quinasas , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-akt , Ratas , Ratas Sprague-Dawley , Sirolimus/farmacología , Serina-Treonina Quinasas TOR , Túnica Íntima/patología , Wortmanina