Thr2446 is a novel mammalian target of rapamycin (mTOR) phosphorylation site regulated by nutrient status

The mammalian target of rapamycin (mTOR) is a key regulator of protein translation. Signaling via mTOR is increased by growth factors but decreased during nutrient deprivation. Previous studies have identified Ser(2448) as a nutrient-regulated phosphorylation site located in the mTOR catalytic domain, insulin stimulates Ser(2448) phosphorylation via protein kinase B (PKB), while Ser(2448) phosphorylation is attenuated with amino acid starvation. Here we have identified Thr(2446) as a novel nutrient-regulated phosphorylation site on mTOR. Thr(2446) becomes phosphorylated when CHO-IR cells are nutrient-deprived, but phosphorylation is reduced by insulin stimulation. Nutrient deprivation activates AMP-activated protein kinase (AMPK). To test whether this could be involved in regulating phoshorylation of mTOR, we treated cultured murine myotubes with 5'-aminoimidazole-4-carboxamide ribonucleoside (AICAR) or dinitrophenol (DNP). Both treatments activated AMPK and also caused a concomitant increase in phosphorylation of Thr(2446) and a parallel decrease in insulin's ability to phosphorylate p70 S6 kinase. In vitro kinase assays using peptides based on the sequence in amino acids 2440-2551 of mTOR found that PKB and AMPK are capable of phosphorylating sites in this region. However, phosphorylation by PKB is restricted when Thr(2446) is mutated to an acidic residue mimicking phosphorylation. Conversely, AMP-kinase-induced phosphorylation is reduced when Ser(2448) is phosphorylated. These data suggest differential phosphorylation Thr(2446) and Ser(2448) could act as a switch mechanism to integrate signals from nutrient status and growth factors to control the regulation of protein translation.