Evidence of an interaction between eIF3f and mTOR. Extracts (750 μl) of 293T cells overexpressing epitope tagged forms of mTOR and eIF3f proteins were incubated with affinity resins or antibodies as described below. After washing resins or immune complexes, the bound proteins were eluted and subjected to SDS–PAGE, along with extract samples (15 μl). Immunoblots were then prepared to detect the epitope tagged proteins. (A) Extracts from cells overexpressing GFP or HA-tagged eIF3f were incubated with GSH-beads (15 μl) bound to either GST (2 μg) or GST-CTmTOR (2 μg). (B) Extracts from 293T cells overexpressing GFP (as a control) or HA-tagged mTOR were incubated with GSH-beads (15 μl) bound to either GST (2 μg) or GST-eIF3f (2 μg). (C) AU1-tagged mTOR and/or HA-tagged eIF3f were overexpressed in 293T cells before immunoprecipitations were conducted with AU1 antibodies. The band appearing just below HA-eIF3f in the AU1 IP is the heavy chain of the AU1 antibody. (D) HA-tagged mTOR and/or FLAG tagged eIF3f were overexpressed before conducting immunoprecipitations with FLAG antibodies.

Identification of eIF4G-1 as a protein that associates with eIF3 in a rapamycin-sensitive manner in response to insulin. 3T3-L1 adipocytes were incubated at 37°C without or with 20 nM rapamycin for 1 h. After adding insulin as indicated, the incubations were continued for 15 min. (A) eIF3 was immunoprecipitated by using antibodies to eIF3b. After thoroughly washing immune complexes, samples were subjected to SDS–PAGE. Proteins were stained with either silver or Coomassie blue. Regions of the gels surrounding eIF3a (which stained very poorly with silver) and eIF3b are shown. The 220K band contains a protein (apparent M_r=220 000) whose abundance increases in response to insulin. (B) A gel slice containing 220K was incubated with trypsin and the resulting peptides were eluted and analyzed by tandem mass spectrometry. Sequences of 18 tryptic peptides were matched to the protein sequence of mouse eIF4G-1 (NP_001005331.1). pS denotes phosphorylated S, which was found in four of the peptides. (C) Extract samples (750 μl) were incubated with protein G-agarose beads to which nonimmune IgG (NI), eIF3b antibodies (eIF3b IP), or eIF4G antibodies (eIF4G IP) had been bound. Immune complexes were washed before immunoprecipitated proteins and extract samples (15 μl) were subjected to SDS–PAGE. Immunoblots of eIF4G, eIF4A, eIF4E, eIF3a, eIF3b, and eIF3i are presented.

Effects of insulin and rapamycin on eIF4G–eIF3 association. (A) Adipocytes were incubated with increasing concentrations of insulin for 15 min. Immune complexes were isolated after incubating extract samples (750 μl) with either eIF3b antibodies (eIF3b IP) or eIF4G antibodies (eIF4G IP). The immunoprecipitated proteins were subjected to SDS–PAGE, and eIF4G and eIF3b immunoblots were prepared. (B) eIF4G and eIF3b that coimmunoprecipitated (CoIP) were expressed relative to the respective maximum amounts recovered. The results are mean values (±s.e.) from three experiments. (C) Adipocytes were incubated with increasing concentrations of rapamycin for 1 h, and then incubated with insulin (10 mU/ml) for 15 min. Immunoblots depict eIF4G and eIF3b recovered in immune complexes isolated using eIF3b antibodies. (D) The amounts of eIF4G that CoIP with eIF3 were expressed as percentages of the eIF4G recovered from cells incubated with insulin alone. The open symbol (○) represents results from cells incubated without insulin or rapamycin. Mean values ±s.e. of three experiments are presented. (E) Adipocytes were incubated without or with 2 nM rapamycin and/or 20 μM FK506 for 1 h. Insulin (10 mU/ml) was then added as indicated, and the incubations were continued for 15 min. Immunoprecipitations were conducted with eIF3b antibodies, and immunoblots were prepared to detect the eIF4G, eIF3b, and eIF4E recovered. Immunoblots of extract samples were prepared with phosphospecific antibodies to sites in 4EBP1, eIF4G, Akt, and S6K1.

The insulin-stimulated, rapamycin-sensitive, association of eIF4G with eIF3 does not require eIF4E binding to eIF4G. (A) FLAG-eIF4E was coexpressed in 293T cells with HA-tagged forms of either wild-type (WT) eIF4G or a mutant eIF4G (AA) rendered deficient in eIF4E binding. eIF4E was partially purified from extract samples (750 μl) by using m⁷GTP agarose. Proteins were eluted from the beads and subjected to SDS–PAGE along with samples (15 μl) of extract before immunoblots were prepared with antibodies to either the HA or FLAG epitopes. (B) Proteins were overexpressed as described above before immunoprecipitations were conducted with FLAG antibodies. Immunoblots were prepared with antibodies to either the HA or FLAG epitopes. (C) HA-tagged eIF4G (WT) and eIF4G (AA) were overexpressed in 3T3-L1 adipocytes by using adenoviral vectors. After 24 h, the cells were incubated without and with rapamycin and/or insulin as described above. Immunoprecipitations were conducted with nonimmune IgG or eIF3b antibodies. Immunoblots were prepared with eIF3b antibodies and antibodies to the HA epitope. (D) The relative amounts of HA-tagged forms of eIF4G that coimmunoprecipitated with eIF3 were expressed as percentages of the maximum HA signal recovered in each experiment. Mean values±s.e. from five experiments are presented.

The insulin-stimulated, rapamycin-sensitive, association of eIF4G with eIF3 does not require eIF3 to be bound to the small ribosomal subunit. Adipocytes were incubated without additions or with insulin and/or rapamycin as described in the legend to Figure 2. (A) Cells were homogenized in PSB, which contained 10 mM KCl, 1.3 mM magnesium acetate, 0.5 mM DTT, and 10 mM HEPES, pH 7.4 (Helentjaris and Ehrenfeld, 1978). The homogenates were centrifuged at 10 000 g for 10 min, and the supernatants (Extract) were removed. Samples of the extract were centrifuged at 174 000 g for 60 min. The resulting supernatants (174S) were collected, and the pellets were suspended in PSB plus 0.5 M KCl. After mixing at 4°C for 20 min, the suspensions were centrifuged at 174 000 g for 60 min. The supernatants (HSE) were collected and pellets (SWR) were suspended in PSB plus 0.1 M KCl. After adjusting the concentration of KCl in the Extract, 174S, and HSE fractions to 0.1 M, immunoprecipitations were conducted using eIF3b antibodies. Immunoblots show eIF4G, eIF3b and rpS6 recovered in the immune complexes. (B) Extracts were prepared as described in Materials and methods before immunoprecipitations were conducted using eIF3b antibodies. Results represent the relative amounts of rpS6 and eIF4G coimmunoprecipitating with eIF3 and are means ±s.e. from five experiments. (C) Adipocyte extracts were layered on sucrose gradients and centrifuged. Immunoblots of eIF4G, eIF3b and rpS6 in fractions from the top of the gradient to the fraction containing 80S ribosomes, and immunoblots of eIF4G that coimmunoprecipitated with eIF3, are presented. The relative amounts of eIF4G coimmunoprecipitating with eIF3 from control (○) and insulin-treated (•) samples are plotted. Positions of the 40S, 60S and 80S ribosomes are indicated by the peaks of absorbance at 254 nm.

Insulin-stimulated dissociation of eIF4B from the eIF3 complex. Adipocytes were incubated at 37°C without or with insulin for 15 min. (A) eIF3 was immunoprecipitated by using antibodies to eIF3b. The region of a silver-stained gel surrounding a protein (80K) that was decreased in response to insulin is shown. (B) A gel slice containing 80K was incubated with trypsin and the resulting peptides were analyzed by mass spectrometry. The sequences of three tryptic peptides unambiguously identifying 80K as mouse eIF4B (AK045250) are shown. (C) Adipocytes were incubated with additions as indicated for 15 min at 37°C. Amino acids were a combination of 2 × MEM mixture plus 0.5 mM leucine. Immunoblots of eIF4G, eIF3b, and eIF4B recovered after performing immunoprecipitations with nonimmune IgG (NI) or eIF3b antibodies are shown. (D) HA-tagged forms of eIF4G, eIF4B, and S6K1 were overexpressed in 293T cells. After immunoprecipitating eIF3 by using eIF3b antibodies, samples of the extracts and immunoprecipitated proteins were subjected to SDS–PAGE. An immunoblot showing HA-eIF4G, HA-eIF4B, and HA-S6K1 is presented, along with an immunoblot of rpS6.

Time courses of insulin responses. 3T3-L1 adipocytes were incubated for increasing times with a maximally effective concentration of insulin (10 mU/ml) before the cells were homogenized. (A) Immunoprecipitations were conducted using extracts (750 μl) and eIF3b antibodies. The immunoprecipitated proteins and extract samples (15 μl) were subjected to SDS–PAGE. Immunoblots were prepared to detect eIF4G, eIF3b, eIF4B, and ribosomal protein S6 (rpS6) that were recovered in the eIF3b immunoprecipitation (eIF3b IP). Phosphospecific antibodies were used to detect changes in phosphorylation of sites in 4EBP1, eIF4G, S6K1, Akt, and the ERK1 and ERK2 isoforms of MAP kinase. (B) The relative amounts of eIF4G that coimmunoprecipitated with eIF3, and the levels of phosphorylated Ser1108 in eIF4G, were determined. (C) The effects of insulin on the phosphorylation of S6K1 and Akt were assessed by immunoblotting with phosphospecific antibodies. The results in (B) and (C) are expressed relative to the respective maximum effects, and are mean values ±s.e. of three experiments. Error bars not shown fall within the symbols.

Effects of inhibitors and activators of PI 3 kinase, mTOR, and MAP kinase signaling on the association of eIF3 and eIF4G. (A) Adipocytes were incubated without inhibitors (None) or with 50 μM LY294002 (LY), 100 nM wortmannin (Wm), 5 mM theophylline (Theo), 20 nM rapamycin (Rap), or 20 μM U0126 (U0). After adding insulin (10 mU/ml) as indicated, the incubations were continued for 15 min. Immunoblots were prepared to detect eIF4G and eIF3b in immune complexes isolated using eIF3b antibodies. (B) The amounts of eIF4G coimmunoprecipitating with eIF3 were expressed relative to the eIF4G recovered from cells that had been incubated with insulin alone. Mean values ±s.e. of three experiments are presented. (C, D) Adipocytes were incubated for 5 min at 37°C without additions (None) or with 10 mU/ml insulin (INS), 5 nM platelet-derived growth factor BB (PDGF), 5 nM insulin-like growth factor 1 (IGF), 5 nM epidermal growth factor (EGF), 5 nM fibroblast growth factor 1 (FGF), 100 nM phorbol 12-myristate 13-acetate (PMA), or 2 × MEM amino acids plus 0.5 mM L-leucine (AA). (C) Immunoblots were prepared using phosphospecific antibodies to the T389 site in S6K1 or the activating sites in ERK1 and ERK2. (D) The relative amounts of eIF4G coimmunoprecipitating with eIF3 were expressed as percentages of the maximum recovered. Mean values ±s.e. of three experiments are presented.