Investigations into inositol phosphates: the mTOR regulators you’ve never heard of!

Posted by daviskd2 on Friday, January 31, 2025 in News.

Inositol phosphates are a family of small metabolites characterized by their different phosphorylation patterns. Inositol hexakisphosphate (IP₆), the fully phosphorylated form of inositol phosphate, is found in many organisms and is the most abundant inositol phosphate in animal and plant cells. IP₆ serves a wide range of functions including regulation of enzyme activity, mediation of protein-protein binding/oligomerization, and functioning as a structural cofactor. In cryo-electron microscopy and crystallography studies, IP₆ has been shown to cocrystallize with the protein mTOR (mechanistic target of rapamycin). mTOR is a serine/threonine kinase which, along with mLST8 and Raptor, make up the nutrient-sensing mTORC1 (mechanistic target of rapamycin complex 1). IP₆ binds to a highly positive pocket in mTOR formed by the FAT domain and referred to as the I-site. While the FAT domain is known to participate in the conformational changes of kinase activation, contradictory studies have failed to elucidate the exact role played by IP₆ in mTOR kinase activity.

Recent work from collaborators Ray Blind, Lucia Rameh and John York aims to put these debates to rest by investigating the impact of inositol phosphate species on mTOR function, both alone and in the context of mTORC1.

The authors first performed autokinase assays using recombinant N-terminal truncated mTOR by itself or co-expressed with LST8 and Raptor. The proteins were incubated for 1 hour with radiolabeled-ATP in the presence or absence of inositol phosphate species and assessed for phosphate incorporation. IP₆, IP₅, and IP₄ all increased phosphate incorporation into mTOR and mTORC1 but no other inositol phosphate or inositol demonstrated a large effect. Similarly, IP₆, IP₅, and IP₄ also all increased phosphorylation of 4EBP peptides, an mTORC1 substrate, in a concentration-dependent manner. Further, IP₆ was shown to increase the solubility of mTOR. Under normal conditions, only 16% of mTOR remained soluble after 30 minutes at 30°C, but the addition of IP₆ to the mix resulted in 64% of mTOR still in solution after the same amount of time.

To determine how IP₆ affects mTOR activity toward exogenous peptide, the authors next assessed enzyme kinetics with various concentrations of ATP. IP₆ decreased the apparent K_m and increased the V_MAX for mTOR alone, but did not impact kinetics for mTOR in complex with LST8 and Raptor. Specifically, the presence of IP₆ stabilized an active conformation of mTOR with a higher affinity for ATP, resulting in the increased rate of catalysis. As mTORC1 is more stable than mTOR alone, this likely accounts for the difference in IP₆ effects. However, during prolonged reactions (4-16 hours) the presence of IP₆ does increase mTORC1 product formation, indicating IP₆ also stabilizes mTORC1 in an active state, although to lesser effect. Finally, the authors demonstrated that the effects of IP₆ are reversible and IP₆ dissociates from mTOR when diluted out, consistent with the ability of mTOR to dynamically respond to IP₆ levels.

Overall, this work has provided the first comprehensive analysis of inositol phosphate regulation of mTOR and mTORC1, increasing our kinetic understanding of mTOR kinase activity and providing a tool to modulate mTOR/mTORC1 in cells.

Read more about this exciting new work in the Journal of Biological Chemistry! ~ Cameron I. Cohen