title: Leucine induced dephosphorylation of Sestrin2 promotes mTORC1 activation.
abstract: The studies described herein were designed to explore the role of Sestrin2 in mediating the selective action of leucine to activate mTORC1. The results demonstrate that Sestrin2 is a phosphoprotein and that its phosphorylation state is responsive to the availability of leucine, but not other essential amino acids. Moreover, leucine availability-induced alterations in Sestrin2 phosphorylation correlated temporally and dose dependently with the activation state of mTORC1, there being a reciprocal relationship between the degree of phosphorylation of Sestrin2 and the extent of repression of mTORC1. With leucine deprivation, Sestrin2 became more highly phosphorylated and interacted more strongly with proteins of the GATOR2 complex. Notably, in cells lacking the protein kinase ULK1, the activation state of mTORC1 was elevated in leucine-deficient medium, such that the effect of re-addition of the amino acid was blunted. In contrast, overexpression of ULK1 led to hyperphosphorylation of Sestrin2 and enhanced its interaction with GATOR2. Neither rapamycin nor Torin2 had any effect on Sestrin2 phosphorylation, suggesting that leucine deprivation-induced repression of mTORC1 was not responsible for the action of ULK1 on Sestrin2. Mass spectrometry analysis of Sestrin2 revealed three phosphorylation sites that are conserved across mammalian species. Mutation of the three sites to phospho-mimetic amino acids in exogenously expressed Sestrin2 promoted its interaction with GATOR2 and dramatically repressed mTORC1 even in the presence of leucine. Overall, the results support a model in which leucine selectively promotes dephosphorylation of Sestrin2, causing it to dissociate from and thereby activate GATOR2, leading to activation of mTORC1.
authors.affiliation: The Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, PO Box 850, Hershey, PA 17033, United States. Electronic address: skimball@psu.edu.
authors.forename: Scot R
authors.initials: SR
authors.lastname: Kimball
authors.affiliation: The Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, PO Box 850, Hershey, PA 17033, United States.
authors.forename: Bradley S
authors.initials: BS
authors.lastname: Gordon
authors.affiliation: The Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, PO Box 850, Hershey, PA 17033, United States.
authors.forename: Jenna E
authors.initials: JE
authors.lastname: Moyer
authors.affiliation: The Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, PO Box 850, Hershey, PA 17033, United States.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Dennis
authors.affiliation: The Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, PO Box 850, Hershey, PA 17033, United States.
authors.forename: Leonard S
authors.initials: LS
authors.lastname: Jefferson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000071189:Autophagy-Related Protein-1 Homolog / Q000378:metabolism; D004586:Electrophoresis; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D007930:Leucine / Q000494:pharmacology*; D013058:Mass Spectrometry; D000076222:Mechanistic Target of Rapamycin Complex 1; D051379:Mice; D008954:Models, Biological; D046912:Multiprotein Complexes / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*; D013997:Time Factors; D000084622:Sestrins
chemical_list: D000071189:Autophagy-Related Protein-1 Homolog; D007930:Leucine; D000076222:Mechanistic Target of Rapamycin Complex 1; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D058570:TOR Serine-Threonine Kinases; C465398:SESN2 protein, human; D000084622:Sestrins; C112444:Ulk1 protein, mouse
keywords: Amino acids; Mechanistic target of rapamycin; Rag GTPases; Signaling; ULK1