Multiple inositol phosphate species enhance stability of active mTOR.

Mechanistic Target of Rapamycin (mTOR) binds the small metabolite inositol hexakisphosphate (IP 6 ) as shown in structures of mTOR, however it remains unclear if IP 6 , or any other inositol phosphate species, can activate mTOR kinase activity. Here, we show that multiple, exogenously added inositol phosphate species (IP 6 , IP 5 , IP 4 and IP 3 ) can all enhance the ability of mTOR and mTORC1 to auto-phosphorylate and incorporate radiolabeled phosphate into peptide substrates in in vitro kinase reactions. Although IP 6 did not affect the apparent K M of mTORC1 for ATP, monitoring kinase activity over longer reaction times showed increased product formation, suggesting inositol phosphates stabilize an active form of mTORC1 in vitro . The effects of IP 6 on mTOR were reversible, suggesting IP 6 bound to mTOR can be exchanged dynamically with the free solvent. Interestingly, we also observed that IP 6 could alter mTOR solubility and electrophoretic mobility in SDS-PAGE in the presence of manganese, suggesting divalent cations may play a role in inositol phosphate regulation of mTOR. Together, these data suggest for the first time that multiple inositol phosphate species (IP 4 , IP 5 and IP 6 ) can dynamically regulate mTOR and mTORC1 by promoting a stable, active state of the kinase. Our data suggest that studies of the dynamics of inositol phosphate regulation of mTOR are well justified.