Inositol phosphates dynamically enhance stability, solubility, and catalytic activity of mTOR
Lucia E Rameh1, John D York2, Raymond D Blind3
1Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, Alabama, USA; Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, USA; Department of Medicine, Division of Diabetes, Endocrinology and Metabolism, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
Abstract:
Mechanistic 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, 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 (IP6 = IP5 > IP4 >> IP3). IP6 increased the VMAX and decreased the apparent KM of mTOR for ATP. Although IP6 did not affect the apparent KM 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 IP6 on mTOR were reversible, suggesting IP6 bound to mTOR can be exchanged dynamically with the free solvent. Interestingly, we also observed that IP6 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 (IP6, IP5, IP4, and to a lesser extent IP3) 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.
Insights
Inositol phosphates, including inositol hexakisphosphate (IP6), dynamically regulate the mechanistic target of rapamycin (mTOR) kinase. These compounds enhance mTOR activity and promote a more stable, soluble active state, justifying further cellular studies.
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.
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