Previously, we showed that muscarinic acetylcholine receptors (mAChRs) promote global protein biosynthesis in SNU-407 colon cancer cells. However, the molecular mechanisms underlying this event are poorly understood. Here, we asked whether mAChRs modulate the activity of eukaryotic translation elongation factor 2 (eEF2), which controls ribosomal translocation during the peptide elongation step. When SNU-407 cells were treated with the cholinergic agonist carbachol, eEF2 phosphorylation at T56 was decreased in a dose- and time-dependent manner. The muscarinic antagonist atropine almost completely blocked this effect of carbachol, demonstrating that mAChRs specifically regulate eEF2 dephosphorylation. We also investigated the signaling pathways that connect mAChR stimulation to eEF2 dephosphorylation using chemical inhibitors. Treating cells with U0126, a potent MEK1/2 inhibitor, decreased carbachol-stimulated eEF2 dephosphorylation. In contrast, the mTORC1 inhibitor rapamycin did not have a significant effect on eEF2 dephosphorylation. We also found that the protein kinase C (PKC) inhibitor GF109203X substantially reduced eEF2 dephosphorylation. Together, our experimental data indicate that the MEK1/2-ERK1/2 pathway and the PKC pathway, but not the mTORC1-S6K1 pathway, are involved in mAChR-mediated eEF2 dephosphorylation.
