Inositol phosphates dynamically enhance stability, solubility, and catalytic activity of mTOR.

Mechanistic target of rapamycin (mTOR) binds the small metabolite inositol hexakisphosphate (IP) as shown in structures of mTOR; however, it remains unclear if IP, or any other inositol phosphate species, function as an integral structural element(s) or catalytic regulator(s) of mTOR. Here, we show that multiple, exogenously added inositol phosphate species can enhance the ability of mTOR and mechanistic target of rapmycin complex 1 (mTORC1) to phosphorylate itself and peptide substrates in in vitro kinase reactions, with the higher order phosphorylated species being more potent (IP = IP > IP >> IP). IP increased the V and decreased the apparent K of mTOR for ATP. Although IP did not affect the apparent K of mTORC1 for ATP, monitoring kinase activity over longer reaction times showed increased product formation, suggesting inositol phosphates stabilize the active form of mTORC1 in vitro. The effects of IP on mTOR were reversible, suggesting IP bound to mTOR can be exchanged dynamically with the free solvent. Interestingly, we also observed that IP could alter mTOR electrophoretic mobility under denaturing conditions and its solubility in the presence of manganese. Together, these data suggest for the first time that multiple inositol phosphate species (IP, IP, IP, and to a lesser extent IP) can dynamically regulate mTOR and mTORC1 by promoting a stable, more soluble active state of the kinase. Our data suggest that studies of the dynamics of inositol phosphate regulation of mTOR in cells are well justified.