Amino acid activation of TOR pathway decreases GSK3β activity and increases Myc protein levels. (A) Time course of Myc protein accumulation upon AA treatment. Cells were serum-starved overnight and then bathed in AA-free medium for 30 min. AAs were added for the indicated time. Myc protein levels were analyzed by western blotting. (B) AA induced Myc protein accumulation through the activation of TORC1. S2 cells were serum-starved (lane 1) and bathed in AA-free medium for 30 min (lane 2). AAs were added back for 2 h in the absence (lane 3) or in the presence of rapamycin (lane 4). Myc accumulation corresponded to phosphorylation of GSK3β at Ser 9. (C) Inhibition of GSK3β by LiCl limits the ability of AAs to induce Myc protein accumulation. Cells were serum-starved and then bathed in AA-free medium. A solution of LiCl or AA, alone or together was added to the AA-free medium for 60 min. Accumulation of Myc protein correlates with phosphorylation of S6K on Thr 389 and of GSK3β on Ser 9. Of note: the effect of LiCl on Myc accumulation is less evident when cells are bathed in medium lacking AA than that observed when LiCl is added to cells bathed in complete Schneider medium and low serum (compare Myc levels in Figure 2C, lane 2 to that in Figure 1D lane 3). This difference could be explained by the possibility that cells bathed in AA-free medium have a reduced rate of protein synthesis. This could result in a delay of the mechanism that controls ubiquitin-induced Myc protein stability thus accounting for the reduced level of Myc observed when LiCl is added to AA-free medium. (D) Ectopic expression of Rheb and S6K increases Myc protein and correlates with phosphorylation of S6K on Thr 398 and of GSK3β on Ser 9. S2 cells were transfected with plasmids encoding for HA-S6K and MYC-Rheb. (E) S2-tub-Gal4 cells expressing tubulin-Gal4 were transfected to express UAS-HA-Myc, and co-transfected with HA-S6K and MYC-Rheb. Expression of the relative proteins was analyzed by western blotting using the indicated antibodies; actin and GSK3β were used as control loading. AA: amino acid; GSK3β: glycogen synthase kinase 3-beta; LiCl: lithium chloride; Rheb: Ras homolog enriched in brain; S6K: p70-S6 ribosomal protein kinase; TOR: target of rapamycin.