RESUMEN
The 4G family of eukaryotic mRNA translation initiation factors is composed of three members (eIF4GI, eIF4GII, and DAP5). Their specific roles in translation initiation are under intense investigations, but how their respective intracellular amounts are controlled remains poorly understood. Here we show that eIF4GI and eIF4GII exhibit much shorter half-lives than that of DAP5. Both eIF4GI and eIF4GII proteins, but not DAP5, contain computer-predicted PEST motifs in their N-termini conserved across the animal kingdom. They are both sensitive to degradation by the proteasome. Under normal conditions, eIF4GI and eIF4GII are protected from proteasomal destruction through binding to the detoxifying enzyme NQO1 [NAD(P)H:quinone oxidoreductase]. However, when cells are exposed to oxidative stress both eIF4GI and eIF4GII, but not DAP5, are degraded by the proteasome in an N-terminal-dependent manner, and cell viability is more compromised upon silencing of DAP5. These findings indicate that the three eIF4G proteins are differentially regulated by the proteasome and that persistent DAP5 plays a role in cell survival upon oxidative stress.
RESUMEN
Pancreatic cancer cells show varying sensitivity to the anticancer effects of gemcitabine. However, as a chemotherapeutic agent, gemcitabine can cause intolerably high levels of toxicity and patients often develop resistance to the beneficial effects of this drug. Combination studies show that use of gemcitabine with the pro-apoptotic cytokine TRAIL can enhance the inhibition of survival and induction of apoptosis of pancreatic cancer cells. Additionally, following combination treatment there is a dramatic increase in the level of the hypophosphorylated form of the tumour suppressor protein 4E-BP1. This is associated with inhibition of mTOR activity, resulting from caspase-mediated cleavage of the Raptor and Rictor components of mTOR. Use of the pan-caspase inhibitor Z-VAD-FMK indicates that the increase in level of 4E-BP1 is also caspase-mediated. ShRNA-silencing of 4E-BP1 expression renders cells more resistant to cell death induced by the combination treatment. Since the levels of 4E-BP1 are relatively low in untreated pancreatic cancer cells these results suggest that combined therapy with gemcitabine and TRAIL could improve the responsiveness of tumours to treatment by elevating the expression of 4E-BP1.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Antineoplásicos/farmacología , Desoxicitidina/análogos & derivados , Regulación Neoplásica de la Expresión Génica , Fosfoproteínas/genética , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis/efectos de los fármacos , Caspasas/genética , Caspasas/metabolismo , Proteínas de Ciclo Celular , Línea Celular Tumoral , Desoxicitidina/farmacología , Combinación de Medicamentos , Sinergismo Farmacológico , Humanos , Páncreas/efectos de los fármacos , Páncreas/metabolismo , Páncreas/patología , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/metabolismo , Fosforilación , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina/genética , Proteína Asociada al mTOR Insensible a la Rapamicina/metabolismo , Proteína Reguladora Asociada a mTOR/genética , Proteína Reguladora Asociada a mTOR/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Imagen de Lapso de Tiempo , GemcitabinaRESUMEN
The eukaryotic translation initiation factor 4GI (eIF4GI) serves as a central adapter in cap-binding complex assembly. Although eIF4GI has been shown to be sensitive to proteasomal degradation, how the eIF4GI steady-state level is controlled remains unknown. Here, we show that eIF4GI exists in a complex with NAD(P)H quinone-oxydoreductase 1 (NQO1) in cell extracts. Treatment of cells with dicumarol (dicoumarol), a pharmacological inhibitor of NQO1 known to preclude NQO1 binding to its protein partners, provokes eIF4GI degradation by the proteasome. Consistently, the eIF4GI steady-state level also diminishes upon the silencing of NQO1 (by transfection with small interfering RNA), while eIF4GI accumulates upon the overexpression of NQO1 (by transfection with cDNA). We further reveal that treatment of cells with dicumarol frees eIF4GI from mRNA translation initiation complexes due to strong activation of its natural competitor, the translational repressor 4E-BP1. As a consequence of cap-binding complex dissociation and eIF4GI degradation, protein synthesis is dramatically inhibited. Finally, we show that the regulation of eIF4GI stability by the proteasome may be prominent under oxidative stress. Our findings assign NQO1 an original role in the regulation of mRNA translation via the control of eIF4GI stability by the proteasome.