Phosphorylation of Mammalian Target of Rapamycin (mTOR) at Ser-2448 IsMediated by p70S6 Kinase

TLDR

mTOR integrates growth factor and nutrient signals, with the PI3K‑AKT pathway mediating mitogenic input to mTOR. The study tests whether rapamycin blocks serum‑induced Ser‑2448 phosphorylation independently of AKT inhibition. The authors show that p70S6K phosphorylates mTOR at Ser‑2448, that rapamycin‑resistant p70S6K restores this phosphorylation, and that amino‑acid status and p70S6K depletion modulate the modification, establishing p70S6K as a key effector.

Abstract

The mammalian target of rapamycin (mTOR) coordinates cell growth with the growth factor and nutrient/energy status of the cell. The phosphatidylinositol 3-kinase-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 rapamycin-treated 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 mitogen- and nutrient-derived stimuli.