Glucose Regulates mTORC1 Through a Mechanism Involving the Sestrins

Open Access
- Author:
- Sam-Clarke, Mahala
- Graduate Program:
- Biomedical Sciences
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- May 23, 2019
- Committee Members:
- Scot R Kimball, Thesis Advisor/Co-Advisor
Ralph Lauren Keil, Committee Member
Lisa M Shantz, Committee Member
Charles H Lang, Committee Member - Keywords:
- Sestrin
mTOR - Abstract:
- The mammalian target of rapamycin (mTOR) is a protein kinase that forms two distinct complexes referred to as mTOR complexes 1 and 2 (mTORC1 and mTORC2, respectively). mTORC1 controls cellular growth and metabolism. There is a growing interest in understanding the underlying mechanism of the Sestrin family of proteins in regulating mTORC1. The Sestrins are highly conserved, stress-induced proteins, that act as negative regulators of mTORC1. Specifically, recent studies have shown that the Sestrins suppress mTORC1 activity by binding to a heteropentameric complex referred to as GATOR2 (GTPase-activating protein (GAP) activity toward Rags (Ras related GTP binding) 2) that itself acts to repress mTORC1. The interaction of all three Sestrin proteins with GATOR2 occurs in in an amino-acid-dependent manner. Interestingly, a recent study showed that the Rag proteins are required not only for amino acid-induced activation of mTORC1, but also for activation of mTORC1 by glucose, suggesting that glucose may activate mTORC1 through the same pathway utilized by amino acids. Based on these observations, we hypothesized that glucose acts through the Sestrin proteins to activate mTORC1. Initially, we confirmed the results of previous studies and showed that mTORC1 activity is suppressed in cells deprived of leucine and that restoration of leucine to deprived cells restores mTORC1 activity to control values. We also showed that leucine-induced changes in mTORC1 activity require the Sestrins, because in cells lacking all three Sestrin proteins (referred to hereafter as Sestrin triple knockout cells (Sestrin TKO cells)) mTORC1 activity unaffected by changes in leucine availability. We extended the initial studies to show that in wild type cells glucose deprivation suppressed mTORC1 activity and resupplementation with glucose activated mTORC1. However, mTORC1 activity was unaffected by changes in glucose availability in Sestrin TKO cells. In part, leucine activates mTORC1 by promoting Sestrin2 phosphorylation, an event that can be detected as decreased electrophoretic mobility during gel electrophoresis. In agreement with the previous studies, leucine deprivation caused a decrease in Sestrin2 electrophoretic mobility and leucine supplementation increased its mobility. In contrast, Sestrin2 electrophoretic mobility was unaffected by changes in glucose availability. The results suggest that although glucose regulates mTORC1 through a Sestrin-dependent pathway, it does so through a mechanism that is independent of Sestrin2 phosphorylation at the sites regulated by leucine.