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

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Signaling

Background:

• Mechanistic target of rapamycin (mTOR) is a key regulator of cell growth and metabolism.
• While mTOR is known to bind inositol hexakisphosphate (IP6), its precise functional role as a structural or catalytic regulator remains unclear.
• Inositol phosphates (IPs) are diverse signaling molecules with various cellular functions.

Purpose of the Study:

• To investigate the regulatory role of inositol phosphate species on mTOR and mTOR complex 1 (mTORC1) kinase activity.
• To determine the impact of different inositol phosphate species on mTOR's catalytic efficiency and substrate phosphorylation.
• To explore the potential of inositol phosphates in modulating mTOR's structural and functional properties.

Main Methods:

• In vitro kinase assays using purified mTOR and mTORC1 complexes.
• Characterization of kinetic parameters (VMAX, KM) for ATP and peptide substrates.
• Analysis of inositol phosphate effects on protein solubility and electrophoretic mobility.
• Reversibility studies of inositol phosphate binding to mTOR.

Main Results:

• Exogenously added inositol phosphates (IP6, IP5, IP4, IP3) enhance mTOR and mTORC1 kinase activity in vitro, with higher phosphorylation states being more potent.
• IP6 increases VMAX and decreases apparent KM for ATP in mTOR, while stabilizing the active form of mTORC1 over time.
• IP6 binding to mTOR is reversible and affects its electrophoretic mobility and solubility.
• Inositol phosphates promote a more stable and soluble active state of mTORC1.

Conclusions:

• Multiple inositol phosphate species dynamically regulate mTOR and mTORC1 activity.
• Inositol phosphates enhance kinase function by promoting a stable, soluble active conformation.
• These findings support further investigation into the cellular dynamics of inositol phosphate regulation of mTOR.