The mTOR (mammalian target of rapamycin) and GCN2 [eukaryotic initiation factor 2 alpha (eIF2α) kinase 4] pathways play critical roles in integrating the organism?s response to insulin, growth factors, energy status, and nutrient availability, and both pathways play key roles in the regulation of translation initiation and thus major roles in regulation of protein synthesis and growth. These pathways provide an interface between nutrient sensing and the regulation of major metabolic responses and, thus, have widespread significance for organismal health, especially as related to diabetes, obesity and cancer. Our long‐term goal is to define the signaling or cross‐talk mechanisms between the ?amino acid sensor? GCN2 and mTOR pathways and to further elucidate the influence of amino acid supply on protein synthesis and insulin signaling. Cross‐talk between mTOR and GCN2 has been reported in yeast and appears to occur in mammalian cells although this has not been directly investigated to any extent. Our preliminary data strongly supports the hypothesis that GCN2 activation by amino acid deprivation (and presumably the activation of other eIF2αkinases by other types of cell stress) will lead to suppression of mTOR signaling both in tissues of whole animals and in cells in culture.
Our specific aims are (1) to determine if GCN2, eIF2αphosphorylation, and/or an increase in activating transcription factor 4 (ATF4) are (is) necessary and/or sufficient for the regulation of S6K and 4E‐BP phosphorylation state (i.e., dephosphorylation in response to amino acid deprivation, or phosphorylation in response to amino acid addition) and the amount of total 4E‐BP in response to amino acids (i.e., increase in total nonphosphorylated 4E‐BP in response to amino acid deprivation);(2) to determine if the decreased phosphorylation of S6K1 and 4E‐BP1 that is observed in response to amino acid deprivation is due to increased action of a phosphatase, to suppressed activity of a kinase, or to a combination of the two, and to determine if a transcriptional target of the eIF2α/ATF4 integrated stress response pathway is responsible for the regulation of S6K1 and 4E‐BP1 phosphorylation state in response to amino acids;and (3) to determine if insulin signaling is impaired in GCN2(‐/‐)cells (or in cells with inactive mutant forms of eIF2αor GADD34) or GCN2(‐/‐) mice due to sustained phosphorylation of S6K, and to determine if GCN2 activation suppresses mTOR target (i.e., S6K and 4EBP) phosphorylation and improves insulin sensitivity (via removal of the feedback inhibition of S6K on insulin signaling) in intact mice. Studies will be carried out in murine embryonic fibroblasts and human liver‐derived cell lines and in whole mice using a variety of approaches including use of cells or animals with loss of function mutations, nutrient‐modified diets or culture medium, and specific inducers of each signaling pathway.

Public Health Relevance

The mTOR and GCN2 pathways play critical roles in integrating the organism?s response to insulin, growth factors, energy status, and nutrient availability and have widespread significance for organismal health. Exploration of cross‐talk between these two nutrient‐sensing pathways will contribute immensely to our full understanding how nutrient deprivation and other stresses signal to both the eIF2αand mTOR pathways to regulate the normal processes of protein synthesis, growth, and stress resistance;to our understanding of how both signaling pathways influence insulin sensitivity, to our understanding of how dysfunction in these pathways due to nutrient overload or other abnormalities contributes to a wide variety of pathological states, including diabetes, obesity and cancer;and to informed use of drug and nutritional therapies to address these pathological states and improve health.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
High Priority, Short Term Project Award (R56)
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Integrative Nutrition and Metabolic Processes Study Section (INMP)
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Silva, Corinne M
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Cornell University
Schools of Earth Sciences/Natur
United States
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Mazor, Kevin M; Dong, Leiming; Mao, Yuanhui et al. (2018) Effects of single amino acid deficiency on mRNA translation are markedly different for methionine versus leucine. Sci Rep 8:8076
Mazor, Kevin M; Stipanuk, Martha H (2016) GCN2- and eIF2?-phosphorylation-independent, but ATF4-dependent, induction of CARE-containing genes in methionine-deficient cells. Amino Acids 48:2831-2842
Sikalidis, Angelos K; Mazor, Kevin M; Lee, Jeong-In et al. (2014) Upregulation of capacity for glutathione synthesis in response to amino acid deprivation: regulation of glutamate-cysteine ligase subunits. Amino Acids 46:1285-96
Sikalidis, Angelos K; Mazor, Kevin M; Kang, Minji et al. (2013) Total 4EBP1 Is Elevated in Liver of Rats in Response to Low Sulfur Amino Acid Intake. J Amino Acids 2013:864757