TOR is a serine/threonine protein kinase with a large molecular size near 300 kDa that belongs to the phosphatidylinositol kinase-related kinase (PIKK) family. The activity of TOR is inhibited under nutrient starvation, which has been known as a crucial step for autophagy induction in eukaryotes [1
]. TOR was firstly described in 1991 as a target protein of the anti-fungal and immunosuppressant agent rapamycin [3
]. Subsequent studies revealed many important functions of TOR in yeast and higher eukaryotes, making the protein kinase to gain its central position in the signaling network that regulates cell growth. The function of TOR in yeast and higher eukaryotes encompasses regulation of translation, metabolism, and transcription in response to nutrients and growth factors. The broad cellular functions of TOR have made the protein kinase to be an important subject for study of cancer, metabolism, longevity, and neurodegenerative diseases.
In yeast, TOR activity is regulated by the availability of nutrients such as nitrogen and carbon [4
]. Similarly, mTOR activity is regulated by amino acid and glucose levels in mammalian cells [5
]. The knowledge on the regulation of TOR has greatly increased since the discoveries of TOR binding partners. Saccharamyces cerevisiae
contains two TOR proteins, TOR1 and TOR2. Both TOR proteins bind KOG1, LST8, and TCO89, whereas TOR2 also binds LST8, Avo1, Avo2, Avo3, and BIT61 to form a separate protein complex [4
]. Similar to yeast TOR, mTOR binds several proteins to form two distinct protein complexes (). mTORC1 (mTOR complex 1) contains raptor (KOG1 ortholog), GβL/mLst8, PRAS40 and DEPTOR, whereas mTORC2 (mTOR complex 2) contains rictor (Avo3 ortholog), GβL/mLst8, Sin1 (Avo1 ortholog), PRR5/protor and DEPTOR [8
Figure 1 Regulation of the mTOR pathway by nutrients (amino acids, glucose), stress and insulin/IGF-1. (a) Two mTOR complexes, mTORC1 and mTORC2, and their components. mTORC1 is the protein complex responsible for autophagy induction in response to nutrient starvation, (more ...)
The yeast TOR complex TORC1, which contains KOG1, and the mammalian mTORC1 containing raptor are key regulators of translation and ribosome biogenesis in yeast and mammalian cells respectively, and they are responsible for autophagy induction in response to starvation. On the other hand, mTORC2 (mTOR-rictor complex), originally known as rapamycin insensitive but likely also targeted by rapamycin, is involved in the regulation of phosphorylation and activation of Akt/PKB, protein kinse C, serum- and glucocorticoid-induced protein kinase 1 [16
]. Because Akt positively regulates mTORC1, it would be reasonable to speculate that mTORC2 acts as a negative regulator of autophagy. Indeed, mTORC2 inhibition induced autophagy and atrophy in skeletal muscle cells under a fasting condition [19
]. However, the autophagy induction by mTORC2 inhibition is mediated mainly by FoxO3, a transcription factor downstream of Akt, that is involved in autophagy gene expression (). Although mTORC2 is involved in autophagy regulation as such, this review is mainly focused on mTORC1-dependent autophagy regulation that is responsive to nutrient starvation.
Availability of cellular amino acids, especially branched chain amino acids such as leucine, regulates mTOR activity (). Amino acid signaling is mediated by the RagA family small GTPases in mammalian cells and Drosophila [21
]. Similarly, in budding yeast the EGO complex containing ras-related GTPases Gtr1 and Gtr2, which belong to the RagA family, and at least two other proteins Ego1 and Ego3 plays important roles in TOR regulation in response to amino acid starvation [23
]. These molecules were initially known to function on the membrane of vacuoles in yeast to regulate growth and microautophagy [23
], implying that the interface between growth and autophagy may occur on the vacoular membrane in yeast [23
]. Another important link of nutrient signalling to mTORC1 involves MAP4K3 (mitogen-activated protein kinase kinase kinase kinase 3) in mammalian cells that is evolutionarily conserved and belongs to the Ste20 family protein kinase [24
]. The kinase activity of MAP4K3 is regulated by amino acid (leucine) levels, indicating that the kinase might act upstream of mTORC1.
What upstream elements regulate Rag GTPases and MAP4K3 and how they functionally interact in mammalian cells remain unclear. Amino acid signaling upstream of these components may start at the surface of cells where the initial contact between amino acids and cells takes place. Amino acid uptake at the plasma membrane is performed by a group of membrane transport proteins classified into a family of solute-linked carrier (SLC) proteins. A recent study revealed that cellular uptake of L-glutamine by SLC1A5 and its subsequent rapid efflux by SLC7A5/SLC3A2, a bidirectional transporter that regulates the simultaneous efflux of L-glutamine and transport of leucine, are important for mTORC1 activation [25
] (). The study showed that loss of SLC1A5 function inhibits cell growth and activates autophagy presumably due to inhibition of tranport of leucine into cells. This transport machinery provides an insight into its potential functional link to the EGO complex in yeast given the role of the EGO complex in the regulation of the general amino acid transporter GAP1 and TORC1 in response to glutamine on the vacuolar surface [23
Another important element in the nutrient-signalling pathway upstream of mTORC1 involves hVps34 (human ortholog of yeast vacuolar protein sorting 34, Vps34, or named PI3KIII (phosphoinositide 3-kinase class III), a lipid kinase conserved throughout eukaryotes. In budding yeast, Vps34 was firstly identified as a component involved in vacuolar sorting [27
]. Later, it was found to regulate autophagy in response to nutrients by producing and accumulating phosphatidylinositol 3-phosphate, PI(3)P, at specific locations during early steps of autophagy induction [28
]. Deficiency of the mammalian hVps34 suppressed leucine-responsive activation of mTORC1, suggesting that hVps34 may act upstream of mTORC1 signaling [29
]. In line with this finding, calcium and calmodulin dependent signaling was shown to regulate mTORC1 via hVps34 in response to leucine in HeLa cells [31
]. However, hVps34 and mTORC1 seem to be in a complicated relationship since Vps34 is not required for TORC1 signalling in Drosophila
]. Perhaps, the complex relation might be due to the difference between the mammalian and Drosophila systems. Alternatively, it could be due to the involvement of hVps34 into multiple different complexes [33
]. It is possible that some of the hVps34 complexes function upstream and others do downstream of mTORC1. Further investigation is necessary to clarify the relation between mTORC1 and hVps34 complexes and their crosstalk during the whole autophagy processes.
While amino acids or leucine is regarded as a signalling molecule that coordinates cell growth with availability of cellular building blocks, glucose may be engaged in the pathway that coordinates growth not only with the availability of the building blocks but also the cellular energy state. It is noteworthy that the signalling pathway regulated by glucose is different from the one regulated by leucine while both pathways are integrated through mTORC1. Glucose starvation reduces the ratio of ATP and AMP in eukaryotic cells and thereby activates AMPK, the 5'-AMP-activated protein kinase, which is potentiated by LKB1, a protein kinase phosphorylating AMPK [34
] (). As a consequence, the activated AMPK inhibits mTORC1 through phosphorylation and activation of tuberous sclerosis complex 2 (TSC2), a negative regulator of mTORC1 [6
]. Consistent with the negative function of AMPK in mTORC1 signaling, AMPK positively regulates autophagy in mammalian cells [36
A recent study revealed that AMPK can inhibit mTORC1 independently of TSC2 by phosphorylating raptor at Ser863 [37
]. Thus, there are two separate pathways that transmit AMPK signalling to mTORC1 (). Another glucose-sensing pathway has been proposed to involve glyceraldehyde-3-phosphate dehydrogenase (GAPDH) that negatively regulates Rheb-mTORC1 signaling independently of TSC1/2 under low glucose influx [38
]. This GAPDH-dependent pathway has a distinct feature relative to the above two AMPK-dependent pathways given that glycolytic influx, rather than energy state, is a signal that regulates mTORC1 (). Taken together, these studies suggest that glucose could regulate mTORC1 through at least three different pathways. Perhaps, the AMPK-mTORC1 pathways might be responsible for monitoring cellular energy status and stress conditions, whereas the GAPDH-mTORC1 pathway might be responsible for the cellular glucose metabolic status. It is important to note that mTORC1 is a converging point for these three pathways.