NAD(P)H quinone-oxydoreductase 1 protects eukaryotic translation initiation factor 4GI from degradation by the proteasome

Abstract

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.

FIG. 1.

Coimmunoprecipitation of eIF4GI and NQO1. (A) Interaction between endogenous proteins. Extracts from HEK-293 cells were either subjected to immunoprecipitation (IP) or processed directly for SDS-PAGE and Western blotting (WB) with the indicated antibodies. R-20 and C-19 are two goat anti-NQO1 antibodies directed against two distinct NQO1 epitopes. In all lanes, NQO1 was revealed using a mouse monoclonal anti-NQO1 antibody. Data are representative of three separate experiments. (B) Diagram illustrating the different HA-tagged fragments of eIF4GI. The domains of eIF4GI known to bind cellular proteins are depicted. HA-4GI, HA-tagged full-length eIF4GI; Mnk1, mitogen-activated protein kinase (MAPK)-interacting protein kinase 1. The black box represents the HA tag inserted in frame at the N terminus of each eIF4GI construct. (C) Lack of interaction between HA-tagged eIF4GI fragments introduced by transfection and endogenous NQO1. Cos-7 cells were transfected with the indicated plasmids and allowed to grow for 36 h. Extracts were then processed for SDS-PAGE and Western blotting with the indicated antibodies either directly (left) or after immunoprecipitation with anti-HA antibodies (right). Data are representative of two independent experiments. (D) NQO1 interaction with eIF4GI is independent of RNA and does not evict eIF4E and eIF4A. Extracts from HEK-293 cells were immunoprecipitated with eIF4GI (left) or NQO1 (right) antibodies. Immunoprecipitates were incubated with or without an RNase A-RNase T1 mixture and processed for SDS-PAGE and Western blotting with the indicated antibodies. Data are representative of two independent experiments. Numbers to the left of the gels are molecular size markers.

FIG. 2.

Decreased eIF4GI amounts and inhibition of protein synthesis in dicumarol-treated cells. (A) Results of a kinetic study. NIH 3T3 cells were treated with 300 μM dicumarol (Dic) for the indicated times. (Bottom) Cell extracts were resolved by SDS-PAGE, and proteins were revealed by Western blotting with the indicated antibodies; (top) the eIF4GI amount was determined and normalized to the β-actin level as described in Materials and Methods. Data are the means ± SD of results from three independent experiments. (B) ODC activity. NIH 3T3 cells were treated as described above, and ODC activities in cell extracts were monitored as described in Materials and Methods. Data are the means ± SD of results from three separate experiments. (C) Results from a dose-response study. NIH 3T3 cells were treated for 6 h with increasing concentrations of dicumarol, as indicated. (Bottom) Cell extracts were resolved by SDS-PAGE, and proteins were revealed by Western blotting with the indicated antibodies; (top) the eIF4GI amount was determined and normalized to the β-actin level as described in Materials and Methods. Data are representative of two independent experiments. (D) Dicumarol inhibits protein synthesis. NIH 3T3 cells were incubated with a sublethal dose of puromycin (puro) for 20 min, and puromycin incorporation into elongating peptides was visualized by Western blotting using antipuromycin antibodies as described in Materials and Methods. (E) eIF4GI half-life. (Top) NIH 3T3 cells were incubated in the presence of cycloheximide (cycloH), dicumarol, or both for the indicated times, cell extracts were resolved by SDS-PAGE, and proteins were revealed by Western blotting using anti-eIF4GI antibodies. (Bottom) NIH 3T3 cells were incubated in the presence of cycloheximide for the indicated times, cell extracts were resolved by SDS-PAGE, and proteins were revealed by Western blotting with the indicated antibodies. D1, cyclin D1. Data are representative of two independent experiments. Numbers to the left of the gels are molecular size markers.

FIG. 3.

Characterization of eIF4GI degradation fragments. (A) Visualization of degradation fragments. NIH 3T3 cells were treated with 300 μM dicumarol for the indicated times. Cell extracts were resolved by SDS-PAGE, and proteins were revealed by Western blotting with the indicated antibodies. (B) Dicumarol and doxorubicin (Doxo) treatments generate distinct fragments. NIH 3T3 cells were treated with 300 μM dicumarol or with 5 μM doxorubicin for 10 h. Cell extracts were resolved by SDS-PAGE, and proteins were revealed by Western blotting with the indicated antibodies. Numbers to the left of the gels are molecular size markers.

FIG. 4.

Dicumarol-mediated degradation of eIF4GI is dependent on the proteasome. (A) MG-132 and lactacystin (Lacta) produce distinct effects on the steady-state level of eIF4GI. NIH 3T3 cells were treated with 25 μM MG-132 or with 5 μM lactacystin for 10 h. Cell extracts were resolved by SDS-PAGE, and proteins were revealed by Western blotting with the indicated antibodies. cPARP, cleaved PARP; cCasp 3, cleaved caspase 3. (B) Results of a kinetic study with lactacystin. NIH 3T3 cells were treated with 5 μM lactacystin for the indicated times. (Bottom) Cell extracts were resolved by SDS-PAGE, and proteins were revealed by Western blotting with the indicated antibodies; (top) the eIF4GI amount was determined and normalized to the β-tubulin level as described in Materials and Methods. Data are the means ± SD of results from three independent experiments. (C) Lactacystin reverses dicumarol-induced degradation of eIF4GI. NIH 3T3 cells were pretreated or not with 5 μM lactacystin for 10 h and treated or not with 300 μM dicumarol for the indicated times. (Bottom) Cell extracts were resolved by SDS-PAGE, and proteins were revealed by Western blotting with the indicated antibodies; (top) the eIF4GI amount was determined and normalized to the β-actin level as described in Materials and Methods. Data are the means ± SD of results from three independent experiments. Numbers to the left of the gels are molecular size markers.

FIG. 5.

Manipulation of the NQO1 amount affects the eIF4GI steady-state level. (A) Validation of siRNAs. NIH 3T3 cells were transfected with two concentrations of control siRNA (siCTRL) or NQO1 siRNA (siNQO1), as indicated. Following a 48-h period, cell extracts were resolved by SDS-PAGE and proteins were revealed by Western blotting with the indicated antibodies. (B) NQO1 silencing diminishes the eIF4GI amount. NIH 3T3 cells were transfected with 50 nM control siRNA or NQO1 siRNA for the indicated times. (Bottom) Cell extracts were then resolved by SDS-PAGE, and proteins were revealed by Western blotting with the indicated antibodies; (top) the eIF4GI amount was determined and normalized to the β-tubulin level as described in Materials and Methods. Data are the means ± SD of results from three independent experiments. (C) NQO1 overexpression augments the eIF4GI amount. NIH 3T3 cells were untransfected or transfected with increasing concentrations of a plasmid encoding NQO1 as described in Materials and Methods. Transfected cells were let grow for 36 h. (Bottom) Cell extracts were resolved by SDS-PAGE, and proteins were revealed by Western blotting with the indicated antibodies; (top) the eIF4GI amount was determined and normalized to the β-tubulin level as described in Materials and Methods. Data are the means ± SD of results from three separate experiments. (D) NQO1 overexpression protects eIF4GI from dicumarol-induced degradation. Cells were untransfected or transfected with a plasmid encoding NQO1. Transfected cells were let grow for 36 h and treated or not for 6 h with 300 μM dicumarol. Cell extracts were then resolved by SDS-PAGE, and proteins were revealed by Western blotting with the indicated antibodies. Data are representative of two separate experiments. Numbers to the left of the gels are molecular size markers.

FIG. 6.

eIF4GI dissociation from the cap-binding complex precedes degradation. (A) Dicumarol treatment activates 4E-BP1. NIH 3T3 cells were treated with 300 μM dicumarol for the indicated times. Cell extracts were resolved by SDS-PAGE, and proteins were revealed by Western blotting with the indicated antibodies. Results from exposure to X-ray films for two different durations (long and short) for 4E-BP1 Western blotting are shown. Data are representative of three independent experiments. (B) Activated 4E-BP1 evicts eIF4GI from the cap-binding complex. Extracts from cells untreated or treated with 300 μM dicumarol for 2 h were subjected to immunoprecipitation using anti-eIF4E antibodies. Immunoprecipitated proteins were then resolved by SDS-PAGE and revealed by Western blotting with the indicated antibodies. Data are representative of three separate experiments. Numbers to the left of the gels are molecular size markers.

FIG. 7.

Oxidative stress induces eIF4GI degradation by the proteasome. (A) Hydrogen peroxide-dependent degradation of eIF4GI. NIH 3T3 cells were treated with 100 μM H₂O₂ for the indicated times. Cell extracts were resolved by SDS-PAGE, proteins were revealed by Western blotting with the indicated antibodies, and the eIF4GI amount was determined and normalized to the β-tubulin level as described in Materials and Methods. Data are the means ± SD of results from three independent experiments. ODC activities in cell extracts were also monitored as described in Materials and Methods. (B) eIF4GI degradation triggered by H₂O₂ is blocked by lactacystin. Cells were pretreated or not with 5 μM lactacystin for 10 h and treated or not with 50 or 100 μM H₂O₂. (Bottom) Cell extracts were then resolved by SDS-PAGE, and proteins were revealed by Western blotting with the indicated antibodies; (top) the eIF4GI amount was determined and normalized to the β-tubulin level as described in Materials and Methods. Data are the means ± SD of results from three independent experiments. Numbers to the left of the gels are molecular size markers.