Fig. 1.
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 HEK293eIF4G-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.