Following the pioneering studies in the early 1990s that identified the TOR kinases as targets of rapamycin in yeast, experiments in mammalian cells and other model systems have defined several important regulatory steps and components that link TOR activity to upstream signals. Key amongst these regulatory factors are the small GTPase Rheb, which directly associates with TOR to stimulate its signaling activity, and the TSC1 and TSC2 proteins, which together inhibit Rheb through the GAP activity of TSC2. This TSC-Rheb-TOR trio has emerged as a core element of the pathway in metazoans, with the TSC1–TSC2 complex in particular acting as a convergence point for multiple signals that govern TOR. For example, factors that respond to cellular energy level (AMP-activated protein kinase, AMPK), growth factor signaling (Akt) or oxygen status (Redd1) have been shown to regulate TOR signaling through distinct, direct effects on TSC2 activity (reviewed in [4
Although this model is consistent with many experimental findings, it is now clear that some signals regulate TOR by other means. Perhaps most significantly, disruption of TSC genes does not prevent regulation of TOR activity by nutrient status [5
], indicating that this conserved input must occur through an alternate mechanism. In addition, replacement of wild type Tsc1 and Tsc2 genes with mutant copies that lack Akt-dependent phosphorylation sites does not affect growth or viability in Drosophila [6
]. These and other results have motivated efforts to identify additional regulatory mechanisms and factors acting upstream of TOR.
The hunt for nutrient-dependent signaling mechanisms has turned up a number of new potential regulators of this pathway. As TOR activity is highly sensitive to amino acid levels, particularly those of branched chain amino acids such as leucine, amino acid transporters have been considered as candidate factors involved in nutrient sensing. A number of proteins in the amino acid transporter family appear to act as amino acid sensors and signal transducers; often such factors have low transport activity, and are collectively referred to as transceptors (reviewed in [8
]). In Drosophila, the proton-assisted transporter Path has the characteristics of a transceptor acting upstream of TOR [9
mutants are defective for cell growth and display genetic interactions with TOR. Expression of Path in a heterologous system leads to TOR activation, yet has only minimal transport capacity. In yeast, the general amino acid permease GAP1, which is regulated by TOR at the level of sorting to the plasma membrane, also has an amino acid sensor activity that is separable from its transport function, and is important for nutrient-dependent activation of cAMP-dependent protein kinase (PKA) [10
The Rag family of GTPases has recently been identified as a new link between amino acids and TOR. These proteins function as novel heterodimeric GTPases, with the active dimer containing RagA or B (Gtr1 in yeast) and RagC or D (Gtr2) bound to GTP and GDP, respectively. In yeast, these proteins were previously shown to control exit from rapamycin-induced arrest as part of the EGO complex [11
]. Three studies in mammalian cells, Drosophila and yeast now provide evidence that these GTPases are specific mediators of amino acid signaling to TOR [12*
]. Active Rag and Gtr heterodimers are able to bind and activate TORC1, through direct interactions with raptor, and this association is regulated by amino acid levels but not other inputs into TOR. Amino acids also specifically control the GTP loading of these proteins, and work in yeast identified Vam6 as the major guanine nucleotide exchange factor for Gtr1. In contrast to Rheb, Rag heterodimers do not appear to activate TOR directly; rather, Sabatini and coworkers demonstrated that amino acids and Rag proteins facilitate TOR-Rheb interaction, by mediating the intracellular trafficking of TOR to a Rheb-containing late endocytic compartment. This aspect of Rag/Gtr function may differ between yeast and mammalian cells, as GFP fusions to Gtr1, Vam6 and TOR1 co-localize to the vacuolar membrane and endocytic vesicles under both growth and starvation conditions [14*
]. In addition, a metazoan-specific factor, the exocyst regulator RalA (yet another small GTPase), has been implicated in nutrient signaling to TOR downstream of Rheb in HeLa cells [15
]. Nonetheless, together these findings indicate an unexpectedly high degree of conservation in nutrient signaling mechanisms, and underscore the importance of understanding how amino acid levels influence the activity of the Rag GTPases.
A number of other signaling molecules have also been shown to play a role in amino acid-dependent TOR activation. Gulati and colleagues found that amino acids induce an inward flux of Ca2+
in HeLa cells, leading to a calmodulin-dependent activation of the class III PI(3)K hVps34 [16
]. This molecule has previously been reported to promote amino acid signaling to TOR, and has well established functions in the endocytic pathway, consistent with a potential involvement of Vps34 in the endocytic localization of Rheb or TOR. Interestingly, this function does not appear to be conserved in lower eukaryotes [17
]. In other settings, increased cytosolic Ca2+
has been shown instead to suppress TOR signaling and induce autophagy, in a pathway involving Ca2+
/calmodulin-dependent protein kinase-kinase and AMPK [18
]. Finally, the Ste20 kinase ortholog MAP4K3 was recently shown to be required for activation of TOR in response to amino acids in Drosophila S2 cells [19
]. Activity of this protein kinase is also sensitive to amino acid levels.
In addition to these new mechanisms of nutrient signaling, it has become clear that factors such AMPK and Akt can regulate TORC1 more directly than previously appreciated, through additional TSC-independent mechanisms (). For example, AMPK can directly phosphorylate the TORC1 component Raptor, generating a docking site for the inhibitory 14-3-3 protein [20*
]. In a parallel fashion, Akt can phosphorylate PRAS40, a Raptor binding protein that also acts as an inhibitor of TORC1. Akt-mediated phosphorylation of PRAS40 again promotes 14-3-3 binding, in this case leading to relief from PRAS40-mediated inhibition [21
]. Thus Raptor is emerging as an important integration site for TORC1 regulation, in addition to its roles in substrate binding. Indeed, Raptor itself has been shown to act as a substrate for TOR in response to growth factors and amino acids, and phosphorylation of Raptor at S863 was found to be critical for full TORC1 activity [25
]. Finally, inhibition of TOR signaling by hypoxia also signals through TSC-dependent and -independent routes, the latter through the hypoxia-inducible BH3 protein Bnip3, which can directly bind and inhibit GTP loading of Rheb [27
Mechanisms of signaling redundancy in the TOR pathway
Rheb has also recently been shown to have both direct and indirect effects on TORC1 signaling. Sato and colleagues found that the direct, binding-dependent effect of Rheb on TOR does not act on TOR’s intrinsic kinase activity; instead, Rheb stimulates TORC1 signaling by promoting interaction with its substrates [28
]. An indirect effect of Rheb on TORC1 signaling involves Rheb-mediated, TOR-independent activation of phospholipase-D1 (PLD1). PLD1 and its product phosphatidic acid were shown to be necessary and sufficient for TOR activation downstream of Rheb [29
]. Altogether, these findings indicate that TOR’s upstream regulators have evolved multiple, redundant and integrated mechanisms of control over TORC1 activity.