TOR Kinase Research Articles

Targeting TOR dependence in cancer

A challenge in cancer therapy has been to identify targets whose function is essential for survival of malignant cells but not normal cells. This Perspective discusses recent evidence that novel inhibitors of the kinase TOR can provide an unprecedented balance of anti-cancer efficacy and tolerability.

Translation Regulators Mediate the Interaction between Eukaryotic Elongation Factor 1A and the Actin Cytoskeleton in Saccharomyces cerevisiae

Eukaryotic Elongation Factor 1A (eEF1A) is an abundant G‐protein whose primary function is to transport aminoacyl‐tRNA to elongating ribosomes during eukaryotic translation. Additionally, eEF1A has also been identified as an actin‐binding protein. This second property of eEF1A allows it to bind and subsequently bundle filamentous actin into two specific structures: patches and cables. These structures collectively referred to as the actin cytoskeleton, provide the network for intracellular transport and endocytosis in yeast. Evidence has substantiated the eEF1A‐actin interaction; however, regulation of this association has yet to be determined. Two major regulators of translation in yeast are the TOR kinases and Gcn2p. These kinases are activated in response to nutrient and amino acid starvation, respectively, and ultimately lead to decreased translation via blocked initiation. Using strains expressing either an F308L or S405P mutant form of eEF1A, both of which exhibit disorganized actin cytoskeletons and blocked translation initiation, we analyzed the affects of TOR and Gcn2p on the eEF1A‐actin interaction. Our current findings suggest that while eEF1A and TOR appear to promote actin cytoskeleton integrity through two distinct pathways, Gcn2p may be involved in eEF1A's ability to sustain actin organization. This research was supported by the National Institutes of Health.

Positive or Negative Roles of Different Cyclin-Dependent Kinase Pho85-Cyclin Complexes Orchestrate Induction of Autophagy in Saccharomyces cerevisiae

As a major intracellular degradation pathway, autophagy is tightly regulated to prevent cellular dysfunction in all eukaryotic cells. The rapamycin-sensitive Tor kinase complex 1 is a major regulator of autophagy. Several other nutrient-sensory kinases also play critical roles to precisely modulate autophagy; however, the network of regulatory mechanisms remains largely elusive. We used genetic analyses to elucidate the mechanism by which the stress-responsive, cyclin-dependent kinase Pho85 and its corresponding cyclin complexes antagonistically modulate autophagy in Saccharomyces cerevisiae. When complexed with cyclins Pho80 and Pcl5, Pho85 negatively regulates autophagy through downregulating the protein kinase Rim15 and the transcription factors Pho4 and Gcn4. The cyclins Clg1, Pcl1, and Pho80, in concert with Pho85, positively regulate autophagy through promoting the degradation of Sic1, a negative regulator of autophagy that targets Rim15. Our results suggest a model in which Pho85 and its cyclin complexes have opposing roles in autophagy regulation.

Genomic analysis of severe hypersensitivity to hygromycin B reveals linkage to vacuolar defects and new vacuolar gene functions in Saccharomyces cerevisiae

The vacuole of Saccharomyces cerevisiae has been a seminal model for studies of lysosomal trafficking, biogenesis, and function. Several yeast mutants defective in such vacuolar events have been unable to grow at low levels of hygromycin B, an aminoglycoside antibiotic. We hypothesized that such severe hypersensitivity to hygromycin B (hhy) is linked to vacuolar defects and performed a genomic screen for the phenotype using a haploid deletion strain library of non-essential genes. Fourteen HHY genes were initially identified and were subjected to bioinformatics analyses. The uncovered hhy mutants were experimentally characterized with respect to vesicular trafficking, vacuole morphology, and growth under various stress and drug conditions. The combination of bioinformatics analyses and phenotypic characterizations implicate defects in vesicular trafficking, vacuole fusion/fission, or vacuole function in all hhy mutants. The collection was enriched for sensitivity to monensin, indicative of vacuolar trafficking defects. Additionally, all hhy mutants showed severe sensitivities to rapamycin and caffeine, suggestive of TOR kinase pathway defects. Our experimental results also establish a new role in vacuolar and vesicular functions for two genes: PAF1, encoding a RNAP II-associated protein required for expression of cell cycle-regulated genes, and TPD3, encoding the regulatory subunit of protein phosphatase 2A. Thus, our results support linkage between severe hypersensitivity to hygromycin B and vacuolar defects.

Rapamycin Inhibits Yeast Nucleotide Excision Repair Independently of Tor Kinases

The yeast target of rapamycin (Tor) kinases, Tor1 and Tor2, belong to the phosphatidylinositol 3-kinase-related family of proteins, which are involved in the cellular response to DNA damage and changes in nutrient conditions. In contrast to yeast, many eukaryotes possess a single Tor kinase. Regardless of the number of Tor kinases in an organism, two distinct complexes involving Tor proteins exist in eukaryotes, TORC1 and TORC2. The yeast TORC1, containing Tor1 or Tor2, is sensitive to the antibiotic rapamycin. The yeast TORC2 is insensitive to rapamycin. We examined the influence of rapamycin treatment upon yeast transcription-coupled nucleotide excision repair in a gene transcribed by RNA polymerase II. We also examined tor mutants for their ability to perform transcription-coupled repair in the absence or presence of rapamycin. Ostensibly lacking TORC1 and TORC2 function, a tor1tor2(ts) mutant grown at the nonpermissive temperature exhibited similar rates of repair as the wild-type strain. However, repair of both strands in genes decreases in the wild-type strain and the tor1tor2(ts) mutant exposed to rapamycin. Rapamycin may be inhibiting DNA repair independently of the Tor kinases. In yeast, FPR1 encodes the rapamycin-binding protein Fpr1 that inhibits the TORC1 kinase in the presence of rapamycin. Fap1 competes with rapamycin for Fpr1 binding. Deletion of the FPR1 or FAP1 gene abolishes the inhibitory effect of rapamycin on repair. Thus, the decreased repair observed following rapamycin treatment is independent of TORC1/2 function and likely due to a function of Fap1. We suggest that Fap1 and peptidyl-prolyl isomerases, particularly Fpr1, function in the cellular response to genotoxic stress. Our findings have clinical implications for genetic toxicities associated with genotoxic agents when coadministered with rapamycin.

Distinctive Responses to Nitrogen Starvation in the Dominant Active Mutants of the Fission Yeast Rheb GTPase

Rheb, a Ras-like small GTPase conserved from human to yeast, controls Tor kinase and plays a central role in the regulation of cell growth depending on extracellular conditions. Rhb1 (a fission yeast homolog of Rheb) regulates amino acid uptake as well as response to nitrogen starvation. In this study, we generated two mutants, rhb1-DA4 and rhb1-DA8, and characterized them genetically. The V17A mutation within the G1 box defined for the Ras-like GTPases was responsible for rhb1-DA4 and Q52R I76F within the switch II domain for rhb1-DA8. In fission yeast, two events--the induction of the meiosis-initiating gene mei2+ and cell division without cell growth--are a typical response to nitrogen starvation. Under nitrogen-rich conditions, Rheb stimulates Tor kinase, which, in turn, suppresses the response to nitrogen starvation. While amino acid uptake was prevented by both rhb1-DA4 and rhb1-DA8 in a dominant fashion, the response to nitrogen starvation was prevented only by rhb1-DA4. rhb1-DA8 thereby allowed genetic dissection of the Rheb-dependent signaling cascade. We postulate that the signaling cascade may branch below Rhb1 or Tor2 and regulate the amino acid uptake and response to nitrogen starvation independently.

The role of TOG domains in microtubule plus end dynamics

The XMAP215 (Xenopus microtubule-associated protein 215) and CLASP [CLIP-170 (cytoskeletal linker protein 170) associated protein] microtubule plus end tracking families play central roles in the regulation of interphase microtubule dynamics and the proper formation of mitotic spindle architecture and flux. XMAP215 members comprise N-terminally-arrayed hexa-HEAT (huntingtin, elongation factor 3, the PR65/A subunit of protein phosphatase 2A and the lipid kinase Tor) repeats known as TOG (tumour overexpressed gene) domains. Higher eukaryotic XMAP215 members are monomeric and have five TOG domains. Yeast counterparts are dimeric and have two TOG domains. Structure determination of the TOG domain reveals that the six HEAT repeats are aligned to form an oblong scaffold. The TOG domain face composed of intra-HEAT loops forms a contiguous, conserved tubulin-binding surface. Nested within the conserved intra-HEAT loop 1 is an invariant, signature, surface-exposed tryptophan residue that is a prime determinant in the TOG domain-tubulin interaction. The arrayed organization of TOG domains is critical for the processive mechanism of XMAP215, indicative that multiple tubulin/microtubule-binding sites are required for plus end tracking activity. The CLASP family has been annotated as containing a single N-terminal TOG domain. Using XMAP215 TOG domain structure determinants as a metric to analyse CLASP sequence, it is anticipated that CLASP contains two additional cryptic TOGL (TOG-like) domains. The presence of additional TOGL domains implicates CLASP as an ancient XMAP215 relative that uses a similar, multi-TOG-based mechanism to processively track microtubule ends.

Akt Phosphorylates Both Tsc1 and Tsc2 in Drosophila, but Neither Phosphorylation Is Required for Normal Animal Growth

Akt, an essential component of the insulin pathway, is a potent inducer of tissue growth. One of Akt's phosphorylation targets is Tsc2, an inhibitor of the anabolic kinase TOR. This could account for part of Akt's growth promoting activity. Although phosphorylation of Tsc2 by Akt does occur in vivo, and under certain circumstances can lead to reduced Tsc2 activity, the functional significance of this event is unclear since flies lacking Akt phosphorylation sites on Tsc2 are viable and normal in size and growth rate. Since Drosophila Tsc1, the obligate partner of Tsc2, has an Akt phosphorylation motif that is not conserved in mammals, we investigate here whether Akt redundantly phosphorylates the Tsc complex on Tsc1 and Tsc2. We provide evidence that Akt phosphorylates Tsc1 at Ser533. We show that flies lacking Akt phosphorylation sites on Tsc1 alone, or on both Tsc1 and Tsc2 concurrently, are viable and normal in size. This shows that phosphorylation of the Tsc1/2 complex by Akt is not required for Akt to activate TORC1 and to promote tissue growth in Drosophila.

Metabolic control of antifungal drug resistance

Fungi have evolved an elegant repertoire of mechanisms to survive the cellular stress exerted by antifungal drugs such as azoles, which inhibit ergosterol biosynthesis inducing cell membrane stress. The evolution and maintenance of diverse resistance phenotypes is contingent upon cellular circuitry regulated by the molecular chaperone Hsp90 and its client protein calcineurin. Here, we establish a novel role for nutrients and nutrient signaling in azole resistance. The vulnerability of Saccharomyces cerevisiae azole resistance phenotypes to perturbation was contingent upon specific auxotrophies. Using strains that acquired azole resistance by Erg3 loss of function as a model for resistance that depends on cellular stress responses, we delineated genetic and environmental factors that mitigate the translation of genotype into resistance phenotype. Compromising a global regulator that couples growth and metabolism to environmental cues, Tor kinase, provides a powerful strategy to abrogate drug resistance of S. cerevisiae and Candida albicans with broad therapeutic potential.

Tap42-associated protein phosphatase type 2A negatively regulates induction of autophagy

Autophagy is a highly conserved degradative process in eukaryotic cells. This process plays an integral role in cellular physiology, and the levels of autophagy must be precisely controlled to prevent cellular dysfunction. The rapamycin-sensitive Tor kinase complex 1 (TORC1) has a major role in regulating the induction of autophagy; however, the regulatory mechanisms are not fully understood. Here, we find that Tap42 and protein phosphatase type 2A (PP2A) are involved in the regulation of autophagy in yeast. Temperature-sensitive mutant alleles of TAP42 revealed that autophagy was induced without inactivation of TORC1. Absence of the Tap42-interacting protein Tip41 abolished autophagy induction in the tap42 mutants, whereas overexpression of Tip41 activated autophagy. Furthermore, inactivation of PP2A stimulated autophagy and overexpression of a catalytic subunit of PP2A blocked rapamycin-induced autophagy. Our data support a model in which autophagy is negatively regulated by the Tap42-PP2A pathway.

Kinome Sirna Screen Identifies SMG-1 as a Negative Regulator of Hypoxia-inducible Factor-1α in Hypoxia

Hypoxia-inducible factor-1 (HIF-1) plays a central role in tumor progression by regulating genes involved in proliferation, glycolysis, angiogenesis, and metastasis. To improve our understanding of HIF-1 regulation by kinome, we screened a kinase-specific small interference RNA library using a hypoxia-response element (HRE) luciferase reporter assay under hypoxic conditions. This screen determined that depletion of cellular SMG-1 kinase most significantly modified cellular HIF-1 activity in hypoxia. SMG-1 is the newest and least studied member of the phosphoinositide 3-kinase-related kinase family, which consists of ATM, ATR, DNA-PKcs, mTOR, and SMG-1. We individually depleted members of the phosphoinositide 3-kinase-related kinase family, and only SMG-1 deficiency significantly augmented HIF-1 activity in hypoxia. We subsequently discovered that SMG-1 kinase activity was activated by hypoxia, and depletion of SMG-1 up-regulated MAPK activity under low oxygen. Suppressing cellular MAPK by silencing ERK1/2 or by treatment with U0126, a MAPK inhibitor, partially blocked the escalation of HIF-1 activity resulting from SMG-1 deficiency in hypoxic cells. Increased expression of SMG-1 but not kinase-dead SMG-1 effectively inhibited the activity of HIF-1α. In addition, cellular SMG-1 deficiency increased secretion of the HIF-1α-regulated angiogenic factor, vascular epidermal growth factor, and survival factor, carbonic anhydrase IX (CA9), as well as promoted the hypoxic cell motility. Taken together, we discovered that SMG-1 negatively regulated HIF-1α activity in hypoxia, in part through blocking MAPK activation.

Identification of a conserved F-box protein 6 interactor essential for endocytosis and cytokinesis in fission yeast

The F-box domain is a degenerated motif consisting of approximately 40 amino acid residues that specifically bind Skp1, a core component of the SCF (Skp1-Cdc53/Cullin 1-F-box protein) ubiquitin ligase. Recent work, mainly performed in budding yeast, indicates that certain F-box proteins form non-SCF complexes together with Skp1 in the absence of cullins and play various roles in cell cycle and signalling pathways. However, it is not established whether these non-SCF complexes are unique to budding yeast or common in other eukaryotes. In the present paper, using TAP (tandem affinity purification) coupled to MudPIT (Multidimensional Protein Identification Technology) analysis, we have identified a novel conserved protein, Sip1, in fission yeast, as an interacting partner of an essential F-box protein Pof6. Sip1 is a large HEAT (huntingtin, elongation factor 3, the PR65/A subunit of protein phosphatase 2A and the lipid kinase Tor)-repeats containing protein (217 kDa) and forms a complex with Pof6 and Skp1. This complex does not contain cullins, indicating that it is a novel non-SCF complex. Like Pof6 and Skp1, Sip1 is essential for cell viability and temperature-sensitive sip1 mutants display cell division arrest as binucleate cells with septa. Sip1 localizes to the nucleus and dynamic cytoplasmic dots, which are shown in the present study to be endocytic vesicles. Consistent with this, sip1 mutants are defective in endocytosis. Furthermore, towards the end of cytokinesis, constriction of the actomyosin ring and dissociation of type II myosin and septum materials are substantially delayed in the absence of functional Sip1. These results indicate that the conserved Sip1 protein comprises a novel non-SCF F-box complex that plays an essential role in endocytosis, cytokinesis and cell division.

Fission yeast Tor1 functions as part of TORC1 to control mitotic entry through the stress MAPK pathway following nutrient stress

TOR signalling coordinates growth and division to control cell size. Inhibition of Schizosaccharomyces pombe Tor1, in response to a reduction in the quality of the nitrogen source (nutrient stress), promotes mitotic onset through activation of the mitogen-activated protein kinase (MAPK) Sty1 (also known as Spc1). Here we show that ;nutrient starvation' (complete withdrawal of nitrogen or leucine) blocks mitotic commitment by altering Sty1 signalling and that different degrees of Sty1 activation determine these differences in mitotic commitment decisions. Mammals contain one TOR kinase, whereas yeasts contain two. In each case, they comprise two distinct complexes: TORC1 and TORC2. We find that nutrient-stress-induced control of mitotic onset, through Tor1, is regulated through changes in TORC1 signalling. In minimal medium, Tor1 interacts with the TORC1 component Mip1 (raptor), and overexpression of tor1+ generates growth defects reminiscent of TORC1 mutants. Strains lacking the TORC2-specific components Sin1 and Ste20 (rictor) still advance mitotic onset in response to nutrient stress. By contrast, Mip1 and the downstream effector Gad8 (a S6K kinase homologue), like Tor1, are essential for nutrient stress to advance mitotic onset. We conclude that S. pombe Tor1 and Tor2 can both act in TORC1. However, it is the inhibition of Tor1 as part of TORC1 that promotes mitosis following nutrient stress.

An Atg1/Atg13 Complex with Multiple Roles in TOR-mediated Autophagy Regulation

The TOR kinases are conserved negative regulators of autophagy in response to nutrient conditions, but the signaling mechanisms are poorly understood. Here we describe a complex containing the protein kinase Atg1 and the phosphoprotein Atg13 that functions as a critical component of this regulation in Drosophila. We show that knockout of Atg1 or Atg13 results in a similar, selective defect in autophagy in response to TOR inactivation. Atg1 physically interacts with TOR and Atg13 in vivo, and both Atg1 and Atg13 are phosphorylated in a nutrient-, TOR- and Atg1 kinase-dependent manner. In contrast to yeast, phosphorylation of Atg13 is greatest under autophagic conditions and does not preclude Atg1-Atg13 association. Atg13 stimulates both the autophagic activity of Atg1 and its inhibition of cell growth and TOR signaling, in part by disrupting the normal trafficking of TOR. In contrast to the effects of normal Atg13 levels, increased expression of Atg13 inhibits autophagosome expansion and recruitment of Atg8/LC3, potentially by decreasing the stability of Atg1 and facilitating its inhibitory phosphorylation by TOR. Atg1-Atg13 complexes thus function at multiple levels to mediate and adjust nutrient-dependent autophagic signaling.

Breathing lessons: Tor tackles the mitochondria

Breathing lessons: Tor tackles the mitochondria

A small G protein Rhb1 and a GTPase-activating protein Tsc2 involved in nitrogen starvation-induced morphogenesis and cell wall integrity of Candida albicans

Rheb is a new member of the small G proteins of the Ras superfamily in eukaryotic organisms and controls various physiological processes. Activity of Rheb is regulated by Tsc2, a GTPase-activating protein (GAP). In this study, we have identified Candida albicans homologs of Rheb (named as Rhb1) and Tsc2. Deletion of the RHB1 gene showed enhanced sensitivity to rapamycin (an inhibitor of TOR kinase), suggesting that Rhb1 is associated with the TOR signaling pathway in C. albicans. Further analysis indicated RHB1 and TSC2 are involved in nitrogen starvation-induced filamentation, likely by controlling the expression of MEP2 whose gene product is an ammonium permease and a sensor for the nitrogen signal. Moreover, we have demonstrated that Rhb1 is also involved in cell wall integrity pathway, by transferring signals through the TOR kinase and the Mkc1 MAP kinase pathway. Together, this study brings new insights into the complex interplay of signaling and regulatory pathways in C. albicans.

Phosphorylation of the Yeast Nitrate Transporter Ynt1 Is Essential for Delivery to the Plasma Membrane during Nitrogen Limitation

Ynt1 is the sole high affinity nitrate transporter of the yeast Hansenula polymorpha. It is highly regulated by the nitrogen source, by being down-regulated in response to glutamine by repression of the YNT1 gene and Ynt1 ubiquitinylation, endocytosis, and vacuolar degradation. On the contrary, we show that nitrogen limitation stabilizes Ynt1 levels at the plasma membrane, requiring phosphorylation of the transporter. We determined that Ser-246 in the central intracellular loop plays a key role in the phosphorylation of Ynt1 and that the nitrogen permease reactivator 1 kinase (Npr1) is necessary for Ynt1 phosphorylation. Abolition of phosphorylation led Ynt1 to the vacuole by a pep12-dependent end4-independent pathway, which is also dependent on ubiquitinylation, whereas Ynt1 protein lacking ubiquitinylation sites does not follow this pathway. We found that, under nitrogen limitation, Ynt1 phosphorylation is essential for rapid induction of nitrate assimilation genes. Our results suggest that, under nitrogen limitation, phosphorylation prevents Ynt1 delivery from the secretion route to the vacuole, which, aided by reduced ubiquitinylation, accumulates Ynt1 at the plasma membrane. This mechanism could be part of the response that allows nitrate-assimilatory organisms to cope with nitrogen depletion.

Nuclear translocation of Gln3 in response to nutrient signals requires Golgi-to-endosome trafficking in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae has developed specialized mechanisms that enable growth on suboptimal nitrogen sources. Exposure of yeast cells to poor nitrogen sources or treatment with the Tor kinase inhibitor rapamycin elicits activation of Gln3 and transcription of nitrogen catabolite-repressed (NCR) genes whose products function in scavenging and metabolizing nitrogen. Here, we show that mutations in class C and D Vps components, which mediate Golgi-to-endosome vesicle transport, impair nuclear translocation of Gln3, NCR gene activation, and growth in poor nitrogen sources. In nutrient-replete conditions, a significant fraction of Gln3 is peripherally associated with light membranes and partially colocalizes with Vps10-containing foci. These results reveal a role for Golgi-to-endosome vesicular trafficking in TORC1-controlled nuclear translocation of Gln3 and support a model in which Tor-mediated signaling in response to nutrient cues occurs in these compartments. These findings have important implications for nutrient sensing and growth control via mTor pathways in metazoans.

Hsf1 Activation Inhibits Rapamycin Resistance and TOR Signaling in Yeast Revealed by Combined Proteomic and Genetic Analysis

TOR kinases integrate environmental and nutritional signals to regulate cell growth in eukaryotic organisms. Here, we describe results from a study combining quantitative proteomics and comparative expression analysis in the budding yeast, S. cerevisiae, to gain insights into TOR function and regulation. We profiled protein abundance changes under conditions of TOR inhibition by rapamycin treatment, and compared this data to existing expression information for corresponding gene products measured under a variety of conditions in yeast. Among proteins showing abundance changes upon rapamycin treatment, almost 90% of them demonstrated homodirectional (i.e., in similar direction) transcriptomic changes under conditions of heat/oxidative stress. Because the known downstream responses regulated by Tor1/2 did not fully explain the extent of overlap between these two conditions, we tested for novel connections between the major regulators of heat/oxidative stress response and the TOR pathway. Specifically, we hypothesized that activation of regulator(s) of heat/oxidative stress responses phenocopied TOR inhibition and sought to identify these putative TOR inhibitor(s). Among the stress regulators tested, we found that cells (hsf1-R206S, F256S and ssa1-3 ssa2-2) constitutively activated for heat shock transcription factor 1, Hsf1, inhibited rapamycin resistance. Further analysis of the hsf1-R206S, F256S allele revealed that these cells also displayed multiple phenotypes consistent with reduced TOR signaling. Among the multiple Hsf1 targets elevated in hsf1-R206S, F256S cells, deletion of PIR3 and YRO2 suppressed the TOR-regulated phenotypes. In contrast to our observations in cells activated for Hsf1, constitutive activation of other regulators of heat/oxidative stress responses, such as Msn2/4 and Hyr1, did not inhibit TOR signaling. Thus, we propose that activated Hsf1 inhibits rapamycin resistance and TOR signaling via elevated expression of specific target genes in S. cerevisiae. Additionally, these results highlight the value of comparative expression analyses between large-scale proteomic and transcriptomic datasets to reveal new regulatory connections.

TOR Signaling in Fission Yeast

Fission yeast has two TOR kinases, Tor1 and Tor2. Recent studies have indicated that this microbe has a TSC/Rheb/TOR pathway like higher eukaryotes. Two TOR complexes, namely TORC1 and TORC2, have been identified in this yeast, as in budding yeast and mammals. Fission yeast TORC1, which contains Tor2, and TORC2, which contains Tor1, apparently have opposite functions with regard to the promotion of G1 arrest and sexual development. Rapamycin does not inhibit growth of wild-type fission yeast cells, unlike other eukaryotic cells, but precise analyses have revealed that rapamycin affects certain cellular functions involving TOR in this yeast. It appears that fission yeast has a potential to be an ideal model system to investigate the TOR signaling pathways.