TOR Protein Research Articles

Schizosaccharomyces pombe AGC family kinase Gad8p forms a conserved signaling module with TOR and PDK1-like kinases.

The TOR protein is a phosphoinositide kinase-related kinase widely conserved among eukaryotes. Fission yeast tor1 encodes an ortholog of TOR, which is required for sexual development and growth under stressed conditions. We isolated gad8, which encodes a Ser/Thr kinase of the AGC family, as a high-copy suppressor of the sterility of a tor1 mutant. Disruption of gad8 caused phenotypes similar to those of tor1 disruption. Gad8p was less phosphorylated and its kinase activity was undetectable in tor1Delta cells. Three amino acid residues corresponding to conserved phosphorylation sites in the AGC family kinases, namely Thr387 in the activation loop, Ser527 in the turn motif and Ser546 in the hydrophobic motif, were important for the kinase activity of Gad8p. Tor1p was responsible for the phosphorylation of Ser527 and Ser546, whereas Ksg1p, a PDK1-like kinase, appeared to phosphorylate Thr387 directly. Altogether, Tor1p, Ksg1p and Gad8p appear to constitute a signaling module for sexual development and growth under stressed conditions in fission yeast, which resembles the mTOR-PDK1-S6K1 system in mammals and may represent a basic signaling module ubiquitous in eukaryotes.

Multiple Roles of Tap42 in Mediating Rapamycin-Induced Transcriptional Changes in Yeast

Tor proteins, targets of the antiinflammatory drug rapamycin, mediate a conserved signaling pathway required for cell growth and proliferation in eukaryotes. By global transcriptional analysis of Saccharomyces cerevisiae, we have examined the role of the essential protein Tap42 in transcriptional regulation by Tor. We find that Tap42 inactivation, like rapamycin addition, prolongs activation of stress response genes. In contrast, Tap42 inactivation, as does inactivation of the protein phosphatases Sit4 and Pph21/22, blocks rapamycin induction of nitrogen discrimination pathway genes. Tap42 inactivation neither affects ribosomal protein gene expression nor blocks rapamycin-induced repression of these genes. These results indicate that Tap42 can both inhibit and activate protein phosphatases and provide insight into the complex events underlying TOR regulation of transcription.

The Tap42-protein phosphatase type 2A catalytic subunit complex is required for cell cycle-dependent distribution of actin in yeast.

In Saccharomyces cerevisiae, the Tor proteins mediate a wide spectrum of growth-related cellular processes in response to nutrients. The pleiotropic role of the Tor proteins is mediated, at least in part, by type 2A protein phosphatases (PP2A) and 2A-like protein phosphatases. Tor-mediated signaling activity promotes the interaction of phosphatase-interacting protein Tap42 with PP2A and 2A-like protein phosphatases. The distinct complexes formed between Tap42 and different phosphatases mediate various cellular events and modulate phosphorylation levels of many downstream factors in the Tor pathway in a Tor-dependent and rapamycin-sensitive manner. In this study, we demonstrate that the interaction between Tap42 and the catalytic subunits of PP2A (PP2Ac) is required for cell cycle-dependent distribution of actin. We show that mutations in PP2Ac and Tap42 that perturb the interaction cause random distribution of actin during the cell cycle and that overexpression of the Rho2 GTPase suppresses the actin defects associated with the mutants. Our findings suggest that the Tap42-PP2Ac complex regulates the actin cytoskeleton via a Rho GTPase-dependent mechanism. In addition, we provide evidence that PP2A activity plays a negative role in controlling the actin cytoskeleton and, possibly, in regulation of the G(2)/M transition of the cell cycle.

The mTOR Signaling Complex

The protein kinase mTOR phosphorylates S6 kinase (S6K) and eIF-4E binding protein, two regulators of protein synthesis, and an mTOR signaling pathway has been implicated in controlling mammalian cell growth and cell size in response to nutrients and growth factors. The activity of mTOR is controlled in part by its association with raptor (regulatory associated protein of TOR), an interaction that seems to have both positive and negative effects on mTOR activity. For example, overexpression of raptor has been observed to both stimulate and inhibit mTOR activity. A nutrient-sensitive interaction between mTOR and raptor has also been unclear. Kim et al. now report that nutrient-sensitivity of the mTOR-raptor complex requires the interaction of mTOR with another protein called GβL (G protein β subunit-like protein), a widely expressed protein of previously unknown function. An endogenous mTOR-GβL complex was detected in cultured mammalian cells and GβL bound to the kinase domain of mTOR. The mTOR-GβL interaction was more stable, and independent from, mTOR-raptor interaction. GβL appears to be a positive regulator of the mTOR signaling pathway, because overexpression of GβL stimulated mTOR kinase activity and decreased either GβL expression by small interfering RNA or expression of mutants that do not bind mTOR reduced phosphorylation of S6K. In addition, decreased GβL expression reduced raptor association with mTOR, implying that GβL stabilizes the mTOR-raptor complex. Furthermore, the mTOR-raptor complex was only nutrient-sensitive if GβL was expressed. The authors propose opposing actions of GβL and raptor on mTOR in which binding of raptor to a GβL-mTOR complex inhibits GβL-mediated activation of mTOR. Low nutrient conditions promote raptor association with mTOR only when GβL is also docked onto mTOR. Increased nutrients would weaken the raptor-mTOR interaction, allowing GβL to stimulate mTOR. D.-H. Kim, D. D. Sarbassov, S. M. Ali, R. R. Latek, K. V. P. Guntur, H. Erdjument-Bromage, P. Tempst, D. M. Sabatini, GβL, a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR. Mol. Cell 11 , 895-904 (2003). [Online Journal]

Translational control by TOR and TAP42 through dephosphorylation of eIF2alpha kinase GCN2.

Yeast protein kinase GCN2 stimulates the translation of transcriptional activator GCN4 by phosphorylating eIF2alpha in response to amino acid starvation. Kinase activation requires binding of uncharged tRNA to a histidyl tRNA synthetase-related domain in GCN2. Phosphorylation of serine 577 (Ser 577) in GCN2 by another kinase in vivo inhibits GCN2 function in rich medium by reducing tRNA binding activity. We show that rapamycin stimulates eIF2alpha phosphorylation by GCN2, with attendant induction of GCN4 translation, while reducing Ser 577 phosphorylation in nonstarved cells. The alanine 577 (Ala 577) mutation in GCN2 (S577A) dampened the effects of rapamycin on eIF2alpha phosphorylation and GCN4 translation, suggesting that GCN2 activation by rapamycin involves Ser 577 dephosphorylation. Rapamycin regulates the phosphorylation of Ser 577 and eIF2alpha by inhibiting the TOR pathway. Rapamycin-induced dephosphorylation of Ser 577, eIF2alpha phosphorylation, and induction of GCN4 all involve TAP42, a regulator of type 2A-related protein phosphatases. Our results add a new dimension to the regulation of protein synthesis by TOR proteins and demonstrate cross-talk between two major pathways for nutrient control of gene expression in yeast.

Cytoplasmic Compartmentation of Gln3 during Nitrogen Catabolite Repression and the Mechanism of Its Nuclear Localization during Carbon Starvation in Saccharomyces cerevisiae

Regulated intracellular localization of Gln3, the transcriptional activator responsible for nitrogen catabolite repression (NCR)-sensitive transcription, permits Saccharomyces cerevisiae to utilize good nitrogen sources (e.g. glutamine and ammonia) in preference to poor ones (e.g. proline). During nitrogen starvation or growth in medium containing a poor nitrogen source, Gln3 is nuclear and NCR-sensitive transcription is high. However, when cells are grown in excess nitrogen, Gln3 is localized to the cytoplasm with a concomitant decrease in gene expression. Treating cells with the Tor protein inhibitor, rapamycin, mimics nitrogen starvation. Recently, carbon starvation has been reported to cause nuclear localization of Gln3 and increased NCR-sensitive transcription. Here we show that nuclear localization of Gln3 during carbon starvation derives from its indirect effects on nitrogen metabolism, i.e. Gln3 does not move into the nucleus of carbon-starved cells if glutamine rather than ammonia is provided as the nitrogen source. In addition, these studies have clearly shown Gln3 is not uniformly distributed in the cytoplasm, but rather localizes to punctate or tubular structures. Analysis of these images by deconvolution microscopy suggests that Gln3 is concentrated in or associated with a highly structured system in the cytosol, one that is possibly vesicular in nature. This finding may impact significantly on how we view (i) the mechanism by which Tor regulates the intracellular localization of Gln3 and (ii) how proteins move into and out of the nucleus.

Transmitting the signal of excess nitrogen inSaccharomyces cerevisiaefrom the Tor proteins to the GATA factors: connecting the dots

Major advances have recently occurred in our understanding of GATA factor-mediated, nitrogen catabolite repression (NCR)-sensitive gene expression in Saccharomyces cerevisiae. Under nitrogen-rich conditions, the GATA family transcriptional activators, Gln3 and Gat1, form complexes with Ure2, and are localized to the cytoplasm, which decreases NCR-sensitive expression. Under nitrogen-limiting conditions, Gln3 and Gat1 are dephosphorylated, move from the cytoplasm to the nucleus, in wild-type but not rna1 and srp1 mutants, and increase expression of NCR-sensitive genes. 'Induction' of NCR-sensitive gene expression and dephosphorylation of Gln3 (and Ure2 in some laboratories) when cells are treated with rapamycin implicates the Tor1/2 signal transduction pathway in this regulation. Mks1 is posited to be a negative regulator of Ure2, positive regulator of retrograde gene expression and to be itself negatively regulated by Tap42. In addition to Tap42, phosphatases Sit4 and Pph3 are also argued by some to participate in the regulatory pathway. Although a treasure trove of information has recently become available, much remains unknown (and sometimes controversial) with respect to the precise biochemical functions and regulatory pathway connections of Tap42, Sit4, Pph3, Mks1 and Ure2, and how precisely Gln3 and Gat1 are prevented from entering the nucleus. The purpose of this review is to provide background information needed by students and investigators outside of the field to follow and evaluate the rapidly evolving literature in this exciting field.

The TOR-controlled transcription activators GLN3, RTG1, and RTG3 are regulated in response to intracellular levels of glutamine.

The essential, rapamycin-sensitive TOR kinases regulate a diverse set of cell growth-related readouts in response to nutrients. Thus, the yeast TOR proteins function as nutrient sensors, in particular as sensors of nitrogen and possibly carbon. However, the nutrient metabolite(s) that acts upstream of TOR is unknown. We investigated the role of glutamine, a preferred nitrogen source and a key intermediate in yeast nitrogen metabolism, as a possible regulator of TOR. We show that the glutamine synthetase inhibitor L-methionine sulfoximine (MSX) specifically provokes glutamine depletion in yeast cells. MSX-induced glutamine starvation caused nuclear localization and activation of the TOR-inhibited transcription factors GLN3, RTG1, and RTG3, all of which mediate glutamine synthesis. The MSX-induced nuclear localization of GLN3 required the TOR-controlled, type 2A-related phosphatase SIT4. Other TOR-controlled transcription factors, GAT1/NIL1, MSN2, MSN4, and an unknown factor involved in the expression of ribosomal protein genes, were not affected by glutamine starvation. These findings suggest that the TOR pathway senses glutamine. Furthermore, as glutamine starvation affects only a subset of TOR-controlled transcription factors, TOR appears to discriminate between different nutrient conditions to elicit a response appropriate to a given condition.

The Protein Kinase C Pathway Is Required for Viability in Quiescence in Saccharomyces cerevisiae

Protein kinase C, encoded by PKC1, regulates construction of the cell surface in vegetatively growing yeast cells. Pkc1 in part acts by regulating Mpk1, a MAP kinase. Mutants lacking Bck1, a component of the MAP kinase branch of the pathway, fail to respond normally to nitrogen starvation, which causes entry into quiescence. Given that the Tor1 and Tor2 proteins are key inhibitors of entry into quiescence, the Pkc1 pathway may regulate these proteins. We find that pkc1Delta and mpk1Delta mutants rapidly die by cell lysis upon carbon or nitrogen starvation. The Pkc1 pathway does not regulate the TOR proteins: transcriptional changes dependent on inhibition of the TORs occur normally in pkc1Delta and mpk1Delta mutants when starved for nitrogen; pkc1Delta and mpk1Delta mutants die rapidly upon treatment with rapamycin, an inhibitor of the TORs. We find that Mpk1 is transiently activated by rapamycin treatment via a novel mechanism. Finally, we find that rapamycin treatment or nitrogen starvation induces resistance to the cell wall-digesting enzyme zymolyase by a Pkc1-dependent mechanism. Thus, the Pkc1 pathway is not a nutrient sensor but acts downstream of TOR inhibition to maintain cell integrity in quiescence.

The TOR signal transduction cascade controls cellular differentiation in response to nutrients.

Rapamycin binds and inhibits the Tor protein kinases, which function in a nutrient-sensing signal transduction pathway that has been conserved from the yeast Saccharomyces cerevisiae to humans. In yeast cells, the Tor pathway has been implicated in regulating cellular responses to nutrients, including proliferation, translation, transcription, autophagy, and ribosome biogenesis. We report here that rapamycin inhibits pseudohyphal filamentous differentiation of S. cerevisiae in response to nitrogen limitation. Overexpression of Tap42, a protein phosphatase regulatory subunit, restored pseudohyphal growth in cells exposed to rapamycin. The tap42-11 mutation compromised pseudohyphal differentiation and rendered it resistant to rapamycin. Cells lacking the Tap42-regulated protein phosphatase Sit4 exhibited a pseudohyphal growth defect and were markedly hypersensitive to rapamycin. Mutations in other Tap42-regulated phosphatases had no effect on pseudohyphal differentiation. Our findings support a model in which pseudohyphal differentiation is controlled by a nutrient-sensing pathway involving the Tor protein kinases and the Tap42-Sit4 protein phosphatase. Activation of the MAP kinase or cAMP pathways, or mutation of the Sok2 repressor, restored filamentation in rapamycin treated cells, supporting models in which the Tor pathway acts in parallel with these known pathways. Filamentous differentiation of diverse fungi was also blocked by rapamycin, demonstrating that the Tor signaling cascade plays a conserved role in regulating filamentous differentiation in response to nutrients.

Ammonia Regulates VID30 Expression and Vid30p Function Shifts Nitrogen Metabolism toward Glutamate Formation Especially when Saccharomyces cerevisiae Is Grown in Low Concentrations of Ammonia

The GATA family proteins Gln3p and Gat1p mediate nitrogen catabolite repression (NCR)-sensitive transcription in Saccharomyces cerevisiae. When cells are cultured with a good nitrogen source (glutamine, ammonia), Gln3p and Gat1p are restricted to the cytoplasm, whereas with a poor nitrogen source (proline), they localize to the nucleus, bind to the GATA sequences of NCR-sensitive gene promoters, and activate transcription. The target of rapamycin-signaling cascade and Ure2p participate in regulating the cellular localization of Gln3p and Gat1p. Rapamycin, a Tor protein inhibitor, like growth with a poor nitrogen source, promotes nuclear localization of Gln3p and Gat1p. gln3 Delta and ure2 Delta mutants are partially resistant and hypersensitive to growth inhibition by rapamycin, respectively. We show that a vid30 Delta is more rapamycin-sensitive than wild type but less so than a ure2 Delta. VID30 expression is modestly NCR-sensitive, responsive to deletion of URE2, and greatly increases in low ammonia medium. Patterns of gene expression in a vid30 Delta suggest that the Vid30p function shifts the balance of nitrogen metabolism toward the production of glutamate, especially when cells are grown in low ammonia. CAN1, DAL4, DAL5, MEP2, DAL1, DAL80, and GDH3 transcription is down-regulated by Vid30p function with proline as the nitrogen source. An effect, however, that could easily be indirect.

Carbon- and nitrogen-quality signaling to translation are mediated by distinct GATA-type transcription factors.

The target of rapamycin (Tor) proteins sense nutrients and control transcription and translation relevant to cell growth. Treating cells with the immunosuppressant rapamycin leads to the intracellular formation of an Fpr1p-rapamycin-Tor ternary complex that in turn leads to translational down-regulation. A more rapid effect is a rich transcriptional response resembling that when cells are shifted from high- to low-quality carbon or nitrogen sources. This transcriptional response is partly mediated by the nutrient-sensitive transcription factors GLN3 and NIL1 (also named GAT1). Here, we show that these GATA-type transcription factors control transcriptional responses that mediate translation by several means. Four observations highlight upstream roles of GATA-type transcription factors in translation. In their absence, processes caused by rapamycin or poor nutrients are diminished: translation repression, eIF4G protein loss, transcriptional down-regulation of proteins involved in translation, and RNA polymerase I/III activity repression. The Tor proteins preferentially use Gln3p or Nil1p to down-regulate translation in response to low-quality nitrogen or carbon, respectively. Functional consideration of the genes regulated by Gln3p or Nil1p reveals the logic of this differential regulation. Besides integrating control of transcription and translation, these transcription factors constitute branches downstream of the multichannel Tor proteins that can be selectively modulated in response to distinct (carbon- and nitrogen-based) nutrient signals from the environment.

Partitioning the transcriptional program induced by rapamycin among the effectors of the Tor proteins.

In all organisms, nutrients are primary regulators of signaling pathways that control transcription. In Saccharomyces cerevisiae, the Tor proteins regulate the transcription of genes sensitive to the quality of available nitrogen and carbon sources. Formation of a ternary complex of the immunosuppressant rapamycin, its immunophilin receptor Fpr1p and Tor1p or Tor2p results in the nuclear import of several nutrient- and stress-responsive transcription factors. We show that treating yeast cells with rapamycin results in a broader modulation of functionally related gene sets than previously understood. Using chemical epistasis and vector-based global expression analyses, we partition the transcriptional program induced by rapamycin among five effectors (TAP42, MKS1, URE2, GLN3, GAT1) of the Tor proteins, and identify how the quality of carbon and nitrogen sources impinge upon components of the program. Biochemical data measuring Ure2p phosphorylation coupled with the partition analysis indicate that there are distinct signaling branches downstream of the Tor proteins. Whole-genome transcription profiling reveals a striking similarity between shifting to low-quality carbon or nitrogen sources and treatment with rapamycin. These data suggest that the Tor proteins are central sensors of the quality of carbon and nitrogen sources. Depending on which nutrient is limited in quality, the Tor proteins can modulate a given pathway differentially. Integrating the partition analysis of the transcriptional program of rapamycin with the biochemical data, we propose a novel architecture of Tor protein signaling and of the nutrient-response network, including the identification of carbon discrimination and nitrogen discrimination pathways.

HEAT Repeats Mediate Plasma Membrane Localization of Tor2p in Yeast

The subcellular distribution of Tor1p and Tor2p, two phosphatidylinositol kinase homologs and targets of the immunosuppressive drug rapamycin in Saccharomyces cerevisiae, was analyzed. We found that Tor protein is peripherally associated with membranes. Subcellular fractionation and immunofluorescence studies showed that Tor1p and Tor2p associate with the plasma membrane and a second fraction that is distinct from Golgi, vacuoles, mitochondria, and nucleus and may represent vesicular structures. Pulse-chase experiments showed that association of Tor protein with plasma membrane and the second compartment is fast, does not appear to involve components of endocytic, secretory, or Golgi to vacuole transport pathways, and is not affected by the immunosuppressive drug rapamycin. Deletion analysis reveals that two domains within Tor2p independently mediate localization to both compartments. These domains are composed of HEAT repeats that are thought to act as protein-protein interaction surfaces. Our studies therefore place Tor proteins at the site of action of their known downstream effectors and suggest that they may be part of a multiprotein complex.

Tor-mediated induction of autophagy via an Apg1 protein kinase complex.

Autophagy is a membrane trafficking to vacuole/lysosome induced by nutrient starvation. In Saccharomyces cerevisiae, Tor protein, a phosphatidylinositol kinase-related kinase, is involved in the repression of autophagy induction by a largely unknown mechanism. Here, we show that the protein kinase activity of Apg1 is enhanced by starvation or rapamycin treatment. In addition, we have also found that Apg13, which binds to and activates Apg1, is hyperphosphorylated in a Tor-dependent manner, reducing its affinity to Apg1. This Apg1-Apg13 association is required for autophagy, but not for the cytoplasm-to-vacuole targeting (Cvt) pathway, another vesicular transport mechanism in which factors essential for autophagy (Apg proteins) are also employed under vegetative growth conditions. Finally, other Apg1-associating proteins, such as Apg17 and Cvt9, are shown to function specifically in autophagy or the Cvt pathway, respectively, suggesting that the Apg1 complex plays an important role in switching between two distinct vesicular transport systems in a nutrient-dependent manner.

Rapamycin-modulated transcription defines the subset of nutrient-sensitive signaling pathways directly controlled by the Tor proteins.

The immunosuppressant rapamycin inhibits Tor1p and Tor2p (target of rapamycin proteins), ultimately resulting in cellular responses characteristic of nutrient deprivation through a mechanism involving translational arrest. We measured the immediate transcriptional response of yeast grown in rich media and treated with rapamycin to investigate the direct effects of Tor proteins on nutrient-sensitive signaling pathways. The results suggest that Tor proteins directly modulate the glucose activation and nitrogen discrimination pathways and the pathways that respond to the diauxic shift (including glycolysis and the citric acid cycle). Tor proteins do not directly modulate the general amino acid control, nitrogen starvation, or sporulation (in diploid cells) pathways. Poor nitrogen quality activates the nitrogen discrimination pathway, which is controlled by the complex of the transcriptional repressor Ure2p and activator Gln3p. Inhibiting Tor proteins with rapamycin increases the electrophoretic mobility of Ure2p. The work presented here illustrates the coordinated use of genome-based and biochemical approaches to delineate a cellular pathway modulated by the protein target of a small molecule.

TOR kinase homologs function in a signal transduction pathway that is conserved from yeast to mammals

Rapamycin is a natural product with potent antifungal and immunosuppressive activities. Rapamycin binds to the FKBP12 prolyl isomerase, and the resulting protein-drug complex inhibits the TOR kinase homologs. Both the FKBP12 and the TOR proteins are highly conserved from yeast to man, and genetic and biochemical studies reveal that these proteins are the targets of rapamycin in vivo. Treatment of yeast or mammalian cells with rapamycin inhibits translational initiation of a subset of mRNAs and dramatically represses ribosomal mRNA and tRNA transcription. Furthermore, rapamycin exposure blocks cell cycle progression in the early G1 phase of the cell cycle, driving cells into a G0 state and, ultimately, triggering autophagy. Recent findings reveal that the upstream factors regulating the TOR signaling cascade are involved in detecting amino acids, nutrients, or growth factors. These findings indicate that the TOR proteins function in a signal transduction pathway that coordinates nutritional and mitogenic signals to control protein biosynthesis and degradation.

Protein kinase activity and identification of a toxic effector domain of the target of rapamycin TOR proteins in yeast.

In complex with FKBP12, the immunosuppressant rapamycin binds to and inhibits the yeast TOR1 and TOR2 proteins and the mammalian homologue mTOR/FRAP/RAFT1. The TOR proteins promote cell cycle progression in yeast and human cells by regulating translation and polarization of the actin cytoskeleton. A C-terminal domain of the TOR proteins shares identity with protein and lipid kinases, but only one substrate (PHAS-I), and no regulators of the TOR-signaling cascade have been identified. We report here that yeast TOR1 has an intrinsic protein kinase activity capable of phosphorylating PHAS-1, and this activity is abolished by an active site mutation and inhibited by FKBP12-rapamycin or wortmannin. We find that an intact TOR1 kinase domain is essential for TOR1 functions in yeast. Overexpression of a TOR1 kinase-inactive mutant, or of a central region of the TOR proteins distinct from the FRB and kinase domains, was toxic in yeast, and overexpression of wild-type TOR1 suppressed this toxic effect. Expression of the TOR-toxic domain leads to a G1 cell cycle arrest, consistent with an inhibition of TOR function in translation. Overexpression of the PLC1 gene, which encodes the yeast phospholipase C homologue, suppressed growth inhibition by the TOR-toxic domains. In conclusion, our findings identify a toxic effector domain of the TOR proteins that may interact with substrates or regulators of the TOR kinase cascade and that shares sequence identity with other PIK family members, including ATR, Rad3, Mei-41, and ATM.

Mutation of Tyr307 and Leu309 in the protein phosphatase 2A catalytic subunit favors association with the alpha 4 subunit which promotes dephosphorylation of elongation factor-2.

The cellular location and substrate specificity of the catalytic subunit (C) of protein phosphatase 2A (PP2A) depend on its interaction with A and B subunits. The distribution of epitope-tagged wild-type or mutated C subunits was studied by transient expression in COS-7 cells. Wild-type tagged C expressed at low levels formed ABC trimer and AC dimer like the endogenous C. Single mutations of C at the site of phosphorylation (Y307F) or carboxymethylation (L309Q) resulted in recovery of only AC dimer. Double mutation of both residues resulted in association of C with alpha 4 protein (alpha 4), a novel subunit of PP2A, instead of with A and B subunits. Thus, the distribution of C between ABC trimer, AC dimer, and alpha 4C complexes can be affected by modifications of the C-terminal residues. The alpha 4 protein is a homologue of the yeast Tap42 protein that functions downstream of the TOR protein to regulate protein synthesis. Transient overexpression of FLAG-alpha 4 resulted in increased dephosphorylation of elongation factor 2, but had no effect on phosphorylation of either p70S6 kinase or PHAS-I (eIF4E-BP). Signals that affect phosphorylation or methylation of the C subunit of PP2A may promote subunit exchange and direct phosphatase activity to specific intracellular substrates.

Tor proteins and protein phosphatase 2A reciprocally regulate Tap42 in controlling cell growth in yeast.

Tor proteins, homologous to DNA-dependent protein kinases, participate in a signal transduction pathway in yeast that regulates protein synthesis and cell wall expansion in response to nutrient availability. The anti-inflammatory drug rapamycin inhibits yeast cell growth by inhibiting Tor protein signaling. This leads to diminished association of a protein, Tap42, with two different protein phosphatase catalytic subunits; one encoded redundantly by PPH21 and PPH22, and one encoded by SIT4. We show that inactivation of either Cdc55 or Tpd3, which regulate Pph21/22 activity, results in rapamycin resistance and that this resistance correlates with an increased association of Tap42 with Pph21/22. Furthermore, we show Tor-dependent phosphorylation of Tap42 both in vivo and in vitro and that this phosphorylation is rapamycin sensitive. Inactivation of Cdc55 or Tpd3 enhances in vivo phosphorylation of Tap42. We conclude that Tor phosphorylates Tap42 and that phosphorylated Tap42 effectively competes with Cdc55/Tpd3 for binding to the phosphatase 2A catalytic subunit. Furthermore, Cdc55 and Tpd3 promote dephosphorylation of Tap42. Thus, Tor stimulates growth-promoting association of Tap42 with Pph21/22 and Sit4, while Cdc55 and Tpd3 inhibit this association both by direct competition and by dephosphorylation of Tap42. These results establish Tap42 as a target of Tor and add further refinement to the Tor signaling pathway.