Activation of mTOR by inhibitors of translation is dependent on PKC-delta.

4155 TOR, a S/T kinase links environmental conditions to translation initiation in eukaryotic cells. Growth factors activate TOR signaling through the PI3K/Akt pathway, whereas nutritional signals and energy charge regulate mTOR via the AMPK/TSC/Rheb pathway. An important function of mTor is to regulate the protein synthesis via phosphorylating its substrates p70S6K1 and 4EBP1. Here we show that inhibition of global translation rapidly activates mTOR signaling leading of phosphorylation of downstream substrates pathway, suggesting a novel mechanism of mTOR regulation in response to decreased translation. Translational inhibitors, such as anisomycin (AN), cyclohexamade (CHX), and emetine (EME) were used to repress the protein synthesis at different stages (peptide formation, or peptide elongation). Each inhibitor induced a rapid concentration-dependent activation of mTOR as indicated by S6K1 phosphorylation (Thr389) and S6(Ser235/6), suggesting indicating a negative feedback from translational machinery to mTOR. To determine whether activation of mTOR was direct, or through activation upstream we used pharmacologic inhibitors and siRNA to determine where in the PI3K-mTOR pathway translation inhibitors activated signaling to mTOR. API-2, an inhibitor of Akt, did not block AN-induced S6K1/S6 phosphorylation, suggesting that the activation of mTR is independent of Akt. AN still induced S6K1 phosphorylation under conditions that suppressed the TSC/Rheb pathway to mTOR (amino acid deprivation or 2’-deoxyglucose). Further, using Rheb siRNAs, we found that AN still stimulated S6K1/S6 phosphorylation, whereas depletion of Rheb blocked serum or amino acid stimulation of S6K1(pT389) phosphorylation. Therefore, this intracellular signal activated mTOR/S6K1 is independent of AKT/TSC/Rheb. However, the phosphorylation of S6K1 is blocked when cells were treated by PKCδ -specific inhibitor rotterlin, suggesting that PKCδ kinase activity is essential for the activation of mTOR induced by translation inhibition. To test this genetically, siRNA was used to decrease the level of PKCδ (~80%), and blocked AN-induced mTOR activation. Furthermore, we found that PKCδ can phosphorylate mTOR in vitro. Taken together, we identified a novel intracellular signaling cascade emanating from the translational machinery that regulates mTOR activity via PKC δ.