The Saccharomyces cerevisiae
targets of rapamycin, TOR1 and TOR2, are functionally and structurally conserved protein kinases that control a large and diverse set of growth-related readouts in response to nutrient availability (53
). In yeast, rapamycin treatment or TOR depletion results in several physiological changes characteristic of starved cells, including inhibition of translation initiation, inhibition of ribosome biogenesis, specific changes in transcription, sorting and turnover of nutrient permeases, accumulation of storage carbohydrates (such as glycogen), and induction of autophagy (reviewed in references 12
). Nitrogen (in particular glutamine) and possibly carbon are important nutrients in the context of yeast TOR signaling (13
). Mammalian TOR (mTOR) controls translation and other growth-related processes in response to amino acids and a growth factor signal (e.g., insulin). The growth factor input into the mTOR pathway is via phosphatidylinositol 3-kinase (PI3K), PDK1, and protein kinase B (PKB; also known as Akt). The precise nature of the link between mTOR and PI3K signaling remains to be determined but may involve the PKB-mediated phosphorylation and inhibition of the tuberous sclerosis protein complex (TSC1-TSC2) upstream of mTOR. Control of translation by mTOR is via activation of S6 kinase (S6K) and inhibition of the eIF4E inhibitor 4E-BP1 (reviewed in reference 30
The yeast TORs control several readouts via a phosphatase switch composed of the type 2A-related phosphatase SIT4, the PP2A/SIT4-associated protein TAP42, and the TAP42-interacting protein TIP41 (16
). Under good nutrient conditions, TOR promotes the binding of SIT4 to TAP42, thereby maintaining SIT4 inactive. On rapamycin treatment or nutrient depletion (i.e., TOR-inactivating conditions), SIT4 dissociates from its inhibitor TAP42 and is active. Activated SIT4 dephosphorylates and activates several targets, such as the GATA-type transcription factor GLN3, the Ser/Thr kinase NPR1, and TIP41 (2
Under favorable nutrient conditions, TOR globally represses starvation-specific transcription by sequestering several nutrient-responsive transcription factors, such as the GATA factors GLN3 and GAT1, the zinc finger transcription factors MSN2 and MSN4, and the bHLH/Zip factor RTG1/3, in the cytoplasm (12
). In the case of GLN3, TOR prevents the transcription of genes normally induced on nitrogen limitation by promoting the association of GLN3 with the cytoplasmic URE2 protein (2
). The phosphorylation of GLN3, which is antagonized by SIT4, is critical for its interaction with URE2 and thus for its cytoplasmic retention (2
). Similarly, TOR and TAP42 maintain the protein kinase NPR1 in an inactive, phosphorylated state whereas TOR inactivation results in the SIT4-dependent dephosphorylation and activation of NPR1. The phosphorylation state of NPR1, in turn, impinges on the sorting and turnover of amino acid permeases such as the tryptophan permease TAT2 and possibly the general amino acid permease GAP1 (3
). Ultimately, both GLN3 and NPR1 are involved in scavenging or synthesizing alternative nutrient (nitrogen) sources. TOR also signals to the translation machinery via TAP42, but the mechanism by which TAP42 and/or the PP2A phosphatases are involved in the control of protein synthesis is unknown. TOR also negatively regulates RTG1/3, MSN2, and MSN4 but does so independently of SIT4 (2
). For other TOR readouts such as ribosome biogenesis or autophagy, the effector pathways are unknown but may involve novel TOR effectors or cross talk with other growth-controlling signaling pathways.
In addition to its redundant function with TOR1 in a rapamycin-sensitive signaling pathway, TOR2 performs a rapamycin-insensitive function that mediates the polarization of the actin cytoskeleton (53
). Recently, two large TOR complexes comprising the TOR proteins and several partner proteins (KOG1, LST8, and AVO1 to AVO3) have been identified in yeast (38
). At least one of the complexes is conserved in mammals (24
). These structurally and functionally distinct complexes account for the diversity and specificity of TOR signaling in yeast.
The RAS/cyclic AMP (cAMP) pathway in S. cerevisiae
plays a major role in the control of growth and metabolism in response to nutrients. The core of the pathway consists of the guanine nucleotide exchange factor CDC25, which, presumably in response to nutrients, activates the redundant small GTPases RAS1 and RAS2 (RAS). RAS, in turn, activates adenylate cyclase (encoded by the CDC35
gene), resulting in the production of cAMP and activation of protein kinase A (PKA) by dissociation of the PKA regulatory subunit BCY1 and the PKA catalytic subunit(s) (redundantly encoded by the TPK1, TPK2, and TPK3 genes) (7
). The RAS/cAMP pathway negatively regulates cellular physiology characteristic of stationary-phase/nutrient starvation. Thus, cells deficient in RAS/cAMP signaling, similarly to TOR-depleted or rapamycin-treated cells, exhibit a G1
cell cycle arrest, accumulation of storage carbohydrates (e.g., glycogen and trehalose), and specific changes in transcription (7
). RAS/cAMP signaling has also been implicated in the nutrient-mediated control of ribosome biogenesis (reviewed in reference 62
; see Discussion). Consistently, cells with constitutive RAS/cAMP signaling fail to adapt their growth program in response to nutrient starvation and rapidly lose viability. Recently, the complexity of RAS/cAMP signaling has increased as a result of the discovery of a G-protein-coupled receptor system, consisting of GPR1 and its Gα
protein GPA2, that appears to act upstream of adenylate cyclase to stimulate cAMP production, probably in response to glucose (36
). Moreover, cAMP production is a very fine-tuned process that is regulated at different levels involving, for instance, feedback loops and the action of phosphodiesterases (59
). The way in which nutrients are sensed by the RAS/cAMP pathway remains unclear.
The precise mechanism(s) by which PKA controls cell growth is not known. It has long been known that the majority of downstream targets of PKA are enzymes involved in intermediary (carbon) metabolism (59
). More recently, however, other PKA targets have emerged. For instance, PKA negatively regulates the transcription of a large number of stress-responsive genes by phosphorylating and inactivating MSN2 and probably MSN4 (20
). The MSNs are redundant, stress element (STRE, AG4
)-binding transcription activators (6
). The MSNs are also negatively controlled by TOR, via the 14-3-3 proteins BMH1 and BMH2 (2
). Moreover, PKA negatively regulates RIM15, a kinase involved in the control of postdiauxic transcription (48
The RAS/cAMP pathway is connected to other nutrient-regulated signaling components, such as the kinase SCH9 (a yeast PKB homologue) and YAK1/SOK1 (see below) (59
). A functional interaction between the RAS/cAMP pathway and TOR has also been suggested (2
), but this interaction has not been investigated directly and systematically.
Here we report that constitutive activation of the RAS/cAMP pathway suppresses a TOR deficiency. We also provide evidence for regulation of PKA and YAK1 localization by TOR. These findings suggest that the RAS/cAMP pathway is a TOR effector pathway distinct from the previously characterized TAP42/SIT4 pathway.