The mammalian target of rapamycin ( mTOR) coordinates cell growth with the growth factor and nutrient/ energy status of the cell. The phosphatidylinositol 3kinase- AKT pathway is centrally involved in the transmission of mitogenic signals to mTOR. Previous studies have shown that mTOR is a direct substrate for the AKT kinase and identified Ser- 2448 as the AKT target site in mTOR. In this study, we demonstrate that rapamycin, a specific inhibitor of mTOR function, blocks serum- stimulated Ser- 2448 phosphorylation and that this drug effect is not explained by the inhibition of AKT. Furthermore, the phosphorylation of Ser- 2448 was dependent on mTOR kinase activity, suggesting that mTOR itself or a protein kinase downstream from mTOR was responsible for the modification of Ser- 2448. Here we show that p70S6 kinase phosphorylates mTOR at Ser- 2448 in vitro and that ectopic expression of rapamycin- resistant p70S6 kinase restores Ser- 2448 phosphorylation in rapamycintreated cells. In addition, we show that cellular amino acid status, which modulates p70S6 kinase ( S6K1) activity via the TSC/ Rheb pathway, regulates Ser- 2448 phosphorylation. Finally, small interfering RNA- mediated depletion of p70S6 kinase reduces Ser- 2448 phosphorylation in cells. Taken together, these results suggest that p70S6 kinase is a major effector of mTOR phosphorylation at Ser- 2448 in response to both mitogenand nutrient- derived stimuli.
