eIF4G is a substrate for PKCα. (A) Co-IP of PKC isoforms with eIF4G from Tet-induced, TPA-stimulated HEK293^eIF4G-b cells. Serum-starved HEK293^eIF4G-b cells were Tet induced for 24 h and treated for 60 min with DMSO (0) or 200 nM TPA. Cell lysates were subjected to Flag-IP followed by immunoblotting with the indicated antibodies. The experiment was repeated four times with similar results. A representative assay is shown. (B) Immunoblot of eIF4G and its cleavage products with site-specific antibodies. Serum-starved HEK293^eIF4G-b cells were Tet or mock induced for 24 h and treated for 15 min with DMSO (0) or 200 nM TPA. The experiment was repeated twice with similar results. A representative assay is shown. M, molecular mass.

eIF4G(Ser1186) is phosphorylated by PKCα. (A) Optimal substrate consensus for PKCα, sequences of a confirmed substrate (P-glycoprotein of MDR1) (37) and of the proposed phosphorylation site in eIF4G (aa 1179 to 1190). Values in parentheses indicate the relative preference for the amino acids shown (bold letters represent amino acids with values of >1.5). Bold letters in the eIF4G sequence emphasize positions that are equal to the consensus or to the P-glycoprotein phosphorylation site. (B) Identification of the PKCα phosphorylation site in eIF4G. HEK293 cells were transfected for 16 h with CTMC variants or CTN fragments, serum starved for 24 h, and treated for 60 min with DMSO (0) or 200 nM TPA. Cell lysates were subjected to Flag-IP followed by immunoblotting. This assay was repeated three times; a representative experiment is shown. ND, not done.

Identification of eIF4G phosphorylation sites by PKCα in vitro. (A) Time course of phosphorylation of recombinant GST-Ct by purified, recombinant GST-PKCα. This experiment was repeated four times; a representative assay is shown. (B) Sequences of phosphorylated eIF4G peptides determined from tandem mass spectrometry. Bold pS or pT denotes a phosphorylated Ser or Thr residue, respectively. (C) The relative intensities of phosphorylation of corresponding residues in eIF4G were determined by quantitative phospho-proteomic analysis of in vitro phosphorylated, recombinant GST-Ct by purified, recombinant GST-PKCα.

Inhibition of Mnk1 with CGP57380 does not affect the activation of PKCα, Erk1/2, and phosphorylation of eIF4G(Ser1186). (A) Inhibition of eIF4E(Ser209) phosphorylation by CGP57380. Lysates from serum-starved, Tet-induced HEK293^eIF4G-e cells expressing the indicated forms of eIF4G-e were subjected to immunoblotting. The effects of pretreatment with 10 μM CGP57380 and stimulation with 0.5 nM STD are shown. (B) Treatments with CGP57380 and STD-mediated stimulation enhance Mnk1 binding to eIF4G. Serum-starved, Tet-induced HEK293^eIF4G-e cells or the corresponding eIF4G(Ser1186/1188Ala) or eIF4G(Ser1186/1188Glu) mutants were pretreated for 1 h with 10 μM CGP57380 and stimulated with 0.5 nM STD as indicated. Cell lysates were subjected to Flag-IP followed by immunoblotting. Bands were quantified as described in Materials and Methods, and the Mnk1 signal density was adjusted for the signal of the loading control (eIF4E). The Mnk1 signal in mock-induced, untreated cells was set at 1. Comparison of exogenous eIF4G-e variants reveals a potent increase in Mnk1 binding upon STD stimulation in cells pretreated with CGP57380.

eIF4G phosphorylation via PKC-Ras-Erk activation. (A) Schematic representation of signaling pathways and protein kinase inhibitors investigated in this research. TPA/STD-dependent stimulation of PKC activates Mnk1 and eIF4E phosphorylation. (B) Inhibition of TPA-dependent eIF4G phosphorylation by Bis1 and UO126. Tet- or mock-induced, serum-starved HEK293^eIF4G-b cells were pretreated for 1 h with 4 μM Bis1 or 10 μM UO126. Cells were stimulated for 15 min with DMSO (0) or 200 nM TPA. Cell lysates were subjected to immunoblotting (left panel) or Flag-IP followed by immunoblotting (right panel). The experiment was repeated three times with similar results. A representative assay is shown. M, molecular mass. (C) Cell lysates were treated as described in panel B and processed for immunoblotting [eIF4G(Ser1148/1232)] or Flag-IP/immunoblotting [p-substrate (PKC)], followed by quantitative chemiluminescence measurements (see Materials and Methods). The values represent fold increases over untreated cells. The experiment was repeated twice with similar results. A representative assay is shown. (D) Substrate consensus of Erk1/2 (37) compared with the eIF4G sequence (aa 1229 to 1235) around the proposed substrate site at Ser1232. Values in parentheses indicate the relative selectivity for the amino acids. Bold letters in the consensus represent strongly preferred amino acids. Bold letters in the eIF4G sequence indicate positions identical to the consensus.

Mapping the PKCα phosphorylation site in eIF4G with C-terminal truncation variants. (A) Schematic representation of the eIF4G isoforms and C-terminal eIF4G fragments used in this study. All proteins are tagged on either terminus with Myc or Flag tags. CTMCΔMnk1 lacks the C-terminal 15 amino acids conveying Mnk1 binding activity. (B) Phosphorylation of eIF4G(Ser1148/Ser1232) in Tet-induced HEK293^eIF4G-b cells (eIF4G-b) or in HEK293 cells transiently transfected with the CTMC fragment (CTMC). HEK293^eIF4G-b cells were Tet induced and serum starved for 24 h and treated for 60 min with DMSO (0) or 200 nM TPA. HEK293 cells were transfected for 16 h, serum starved for 24 h, and treated for 60 min with DMSO (0) or 200 nM TPA. Cell lysates were subjected to Flag-IP followed by immunoblotting with the indicated antibodies. The experiment was repeated twice, and the results of a representative assay are shown. M, molecular mass. (C) TPA-induced phosphorylation by PKCα, PKCα-binding and Mnk1-binding of CTMC, CTMCΔMnk, CTC or CTM in Tet-induced/transfected HEK293 cells expressing C-terminal eIF4G fragments as indicated. Cells and cell lysates were processed as described for panel B. Experiments described in panels B and C were repeated three times with similar results; representative assays are shown. ND, not done.

Isoform-specific activation of PKCα by STD produces sequential phosphorylation of translation factors. (A) Time course of phosphorylation of PKCα, Erk1/2, and eIF4G(Ser1232) upon TPA or STD activation. Serum-starved, Tet-induced HEK293^eIF4G-e cells were treated for the intervals shown with 200 nM TPA or 0.5 nM STD. Total cell lysates were subjected to SDS-PAGE followed by immunoblotting. (B) Time course of STD-stimulated activation of the PKCα/Raf/Erk pathway and phosphorylation of eIF4G, eIF4E, and S6. Serum-starved, Tet-induced HEK293^eIF4G-e cells were treated for the intervals shown with 0.5 nm STD. Extracts were probed by immunoblotting (left panel) or subjected to Flag-IP and immunoblotting (right panel). (C) Lysates from cells treated as described in panel B were processed for immunoblotting or Flag-IP/immunoblotting and quantitative chemiluminescence measurements. The interval with maximum signal was set at 100%, and the signal intensities at all other time points were calculated in relation to this. This experiment was repeated four times with consistent results; average percent phosphorylation values are shown. Standard deviation values were ≤3% at 2 and 5 min, ≤5% at 15 min, and ≤7% at all other time points.

Kinetic analysis of STD-induced Mnk1 binding to wt eIF4G or its Ser1186/1188 double mutants. (A) Serum-starved, Tet-induced HEK293^eIF4G-e cells or the corresponding eIF4G(Ser1186/1188Ala) and eIF4G(Ser1186/1188Glu) mutants were pretreated for 1 h with 10 μM CGP57380 and stimulated with 0.5 nM STD as indicated. Cell lysates were processed for co-IP of Mnk1 with Flag-IP of exogenous eIF4G. IP of Flag-eIF4G and co-IP of eIF4E are included as controls. This experiment was repeated three times; a representative assay is shown. (B) Chemiluminescence quantification of the kinetics of STD-induced Mnk1-eIF4G binding shown in panel A. The quantification of the kinetics of STD-stimulated eIF4G(Ser1186) phosphorylation reflects data shown in Fig. 5B. The interval with maximum signal was set at 100%, and the signal intensities at all other time points were calculated in relation to this. The data represent average percent phosphorylation values from three independent experiments. Standard deviation values were ≤4% at 0, 2, and 5 min, ≤6% or less at 15 and 90 min, and ≤7% at 45 min. (C) Phosphorylation of Ser1232 in eIF4G occurs upon STD stimulation of cells independent of Ser1186/1188 phosphorylation. IP of Flag-eIF4G-e in serum-starved, Tet-induced HEK293 cells expressing wt eIF4G-e or eIF4G-e(Ser1186/8A). (D) Time course of STD-stimulated Mnk1 co-IP in serum-starved, Tet-induced HEK293 cells expressing the eIF4G-e variants shown. The cells were pretreated for 1 h with 10 μM CGP57380 and stimulated with 0.5 nM STD as indicated. The experiment was repeated twice with similar results. A representative assay is shown.

Proposed schematic model of eIF4G binding to Mnk1 upon PKCα-dependent Ser1186 phosphorylation (22). Linear (A) and three-dimensional (B) domain/motif organization of eIF4G. Mnk1 binding to AA box 2 (gray) and the position of Ser1186 in the interdomain linker of eIF4G are indicated. The three-dimensional structure depicts the mutual orientation of eIF4G/eIF4A and eIF4H structured domains; the interdomain linker is reconstructed based on topology analyses of the helicase complex with eIF4G (22). AA boxes 1 and 2 are represented as filled black and gray circles, respectively. The position of Ser1186 in the interdomain linker suggests a possible role in controlling access to AA box 2 and the Mnk1 binding site in eIF4G. (C) Structure-based sequence alignment of AA box 1 with the eIF4G interdomain linker in the vicinity of the PKCα phosphorylation site (Ser1186; asterisk). Solvent-exposed acidic residues in AA box 1 (gray boxes) and positively charged amino acids in the interdomain linker (open boxes) are indicated. Solvent-exposed aromatic residues Tyr1551 and Trp1564 in AA box 1 (2) are shown in black boxes.