Exploring the landscape of mTOR substrates: identification of two novel targets

TOR (Target of Rapamycin), is a highly conserved serine/threonine protein kinase that plays a central role in regulating cell growth and metabolism. It is activated by nutrients, growth factors, and cellular energy. TOR forms two structurally and functionally distinct complexes, TORC1 and TORC2. TOR signaling activates cell growth, defined as an increase in biomass, by stimulating anabolic metabolism while inhibiting catabolic processes.
With emphasis on mammalian TOR (mTOR), we comprehensively reviewed the literature and identified all reported direct substrates. In the context of recent structural information, we discuss how mTORC1 and mTORC2, despite having a common catalytic subunit, phosphorylate distinct substrates. Based on this analysis we conclude that mTORC1 and mTORC2 phosphorylate a common, minimal motif but rely mostly on distinct binding sequences for substrate recognition. mTORC1, for instance, recognizes some of its substrates via a five amino acid target sequence called TOR signaling (TOS) motif. mTORC1 binding to a TOS peptide facilitates phosphorylation of the target site differently located than the binding site.
Furthermore, by exploiting the aforementioned information, we identified two previously uncharacterized mTOR substrates: LST2 and RTN4. Here we show that LST2, also known as ZFYVE28, contains a TOS motif and is phosphorylated by mTORC1 at serine 670 (S670). Phosphorylation on LST2 S670 by mTORC1 primes monoubiquitination on lysine 87 (K87). Monoubiquitinated and phosphorylated LST2 is stable and display a broad reticular distribution. When mTORC1 is inactive, unphosphorylated LST2 localizes to the endosome and is degraded by the proteasome. Cells deficient in LST2 have higher levels of the epidermal growth factor receptor (EGFR). We therefore propose a mechanism in which mTORC1, via LST2, negatively feeds back on its upstream receptor EGFR in order to maintain signaling homeostasis.
In the second part of this thesis, we searched previously reported phosphorproteomics datasets for the identification of novel mTOR substrates. We found that the ER residential protein reticulon 4 (RTN4) is phosphorylated by mTOR on S15, S107 and S181. Although still preliminary, our data suggest that mTOR-dependent phosphorylation of RTN4 regulates calcium and lipid homeostasis through its interaction with specific partners. RTN4 appears to also regulate the ER-mitochondria associated membranes (MAM) structure. In addition, both RTN4 mRNA and protein levels are increased in a mTOR-driven hepatocellular cancer (HCC) mouse model. Our data suggest that mTOR could regulate ER and mitochondria metabolism through phosphorylation of RTN4.
Collectively, this thesis offers a complete list of all reported mTOR substrates and provides insights into the discovery and characterization of two new mTOR substrates, LST2 and RTN4.