Multiple inositol phosphate species enhance stability of active mTOR

Abstract

SummaryMechanistic Target of Rapamycin (mTOR) binds the small metabolite inositol hexakisphosphate (IP6) as shown in structures of mTOR, however it remains unclear if IP6, or any other inositol phosphate species, can activate mTOR kinase activity. Here, we show that multiple, exogenously added inositol phosphate species (IP6, IP5, IP4and IP3) can all enhance the ability of mTOR and mTORC1 to auto-phosphorylate and incorporate radiolabeled phosphate into peptide substrates inin vitrokinase reactions. Although IP6did not affect the apparent KMof mTORC1 for ATP, monitoring kinase activity over longer reaction times showed increased product formation, suggesting inositol phosphates stabilize an active form of mTORC1in vitro. The effects of IP6on mTOR were reversible, suggesting IP6bound to mTOR can be exchanged dynamically with the free solvent. Interestingly, we also observed that IP6could 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 (IP4, IP5and IP6) 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.