The ability of cells to maintain metabolic homeostasis in response to changes in nutrient availability is critical for cell survival. Autophagy, the major cellular pathway by which macromolecules and organelles are degraded, is upregulated in the face of nutrient starvation and cell stress. The mammalian target of rapamycin complex 1 (mTORC1) integrates various environmental stimuli to regulate cellular processes, including autophagy, cell growth, protein synthesis, and lipid metabolism. Of the various inputs to mTORC1, the amino acid sensing pathway is among the most potent. This dissertation describes three studies that aim to elucidate the molecular mechanisms by which cells regulate autophagy and metabolic homeostasis in response to amino acids.
The first study (Chapter 1) identifies let-7 as a microRNA capable of promoting neuronal autophagy in response to nutrient deprivation by coordinately down-regulating the amino acid sensing pathway to prevent mTORC1 activation. In the course of this work, we identified MAP4K3 as a target of let-7, and found that inhibition of MAP4K3 was sufficient to induce autophagy in neurons.
The second study (Chapter 2) builds upon the MAP4K3 findings from the first study, elucidating MAP4K3 regulation of autophagy via phosphorylation of transcription factor EB (TFEB). In the presence of amino acids, activated MAP4K3 phosphorylates TFEB at serine 3, which is necessary for subsequent mTORC1 phosphorylation of TFEB at serine 211 and sequestration in the cytoplasm by 14-3-3 binding. Loss of MAP4K3 leads to increased TFEB nuclear localization, transcription of TFEB-regulated lysosomal genes, and autophagy induction.
In the final study (Chapter 3), we demonstrate another role for MAP4K3 in autophagy regulation: MAP4K3 regulates both mTORC1 localization and activity via distinct pathways. We hypothesize that MAP4K3 is critical for the activation of mTORC1 through the inhibition of SIRT1, AMPK, and TSC2, upstream inhibitors of mTORC1, in the presence of amino acids. Subsequent to mTORC1 activation, MAP4K3 then regulates the Rag GTPases to mobilize mTORC1 off the lysosome to phosphorylate and activate its substrates.
Through these complex mechanisms, MAP4K3 emerges as a critical regulator of cellular homeostasis in response to amino acids, via both mTORC1 activity and TFEB-mediated autophagy induction.