TOR pathway activation in Zea mays L. tissues: Conserved function between animal and plant kingdoms

In most non-photosynthetic eukaryotes it has been demonstrated a conserved signal transduction pathway, namely TOR-S6K, that coordinates growth and cell proliferation. This pathway targets the translational apparatus to induce selective translation of ribosomal mRNAs as well as stimulate the cell cycle transition through the G₁/S phase. Thus, by activation of this pathway through environmental signals, nutrients, stress, or specific growth factors, such as insulin or insulin-like growth factors (IGF), this pathway allows organisms to regulate growth and cell division. In plants, evidence has shown that TOR protein has been highly conserved through evolution, being involved in growth and cell proliferation control as well. Particularly in maize, a peptide named ZmIGF has been found in actively growing tissues. It targets the maize TOR pathway at the same extent as insulin and, by doing so it induces growth, as well as ribosomal proteins and DNA synthesis. Thus, higher metazoans and plants seem to conserve similar biochemical paths to regulate cell growth through equivalent targets that conduce to activation of the TOR-S6K pathway. Recent research shows evidence that supports this proposal by uncovering the ZmIGF receptor in maize, providing further means for analyzing the role of the conserved TOR signaling pathway in this plant.     

PI3-kinase and TOR: PIKTORing cell growth

Regulation of growth and proliferation in higher eukaryotic cells results from an integration of nutritional, energy, and mitogenic signals. Biochemical processes underlying cell growth and proliferation are governed by the phosphatidylinositol 3-kinase (PI3K) and target of rapamycin (TOR) signaling pathways. The importance of the interplay between these two pathways is underscored by the discovery that the TOR inhibitor rapamycin is effective against tumors caused by misregulation of the PI3K pathway. We review here recent data concerning the convergence of the PI3K and TOR pathways, the role of these pathways in cell growth and proliferation, and the regulation of growth by downstream TOR targets.     

Distinctive expression and functional regulation of the maize (Zea mays L.) TOR kinase ortholog

TOR (Target of rapamycin) kinase is a central component of a signal transduction pathway that regulates cellular growth in response to nutrients, mitogens and growth factors in eukaryotes. Knowledge of the TOR pathway in plants is scarce, and reports in agronomical relevant plants are lacking. Previous studies indicate that Arabidopsis thaliana TOR (AtTOR) activity is resistant to rapamycin whereas maize TOR (ZmTOR) is not, suggesting that plants might have different regulation mechanisms for this signal transduction pathway. In the present work maize ZmTOR cDNA was identified and its expression regulation was analyzed during germination on different tissues at various stages of differentiation and by the main ZmTOR regulators. Our results show that ZmTOR contains all functional domains characteristic of metazoan TOR kinase. ZmTOR expression is highly regulated during germination, a critical plant development period, but not on other tissues of contrasting physiological characteristics. Bioinformatic analyses indicated that maize FKBP12 and rapamycin form a functional structure capable of targeting the ZmTOR protein, similar to other non-plant eukaryotes, further supporting its regulation by rapamycin (in contrast with the rapamycin insensitivity of Arabidopsis thaliana) and the conservation of rapamycin regulation through plant evolution.     

Regulation of chemotaxis by the orchestrated activation of Ras, PI3K, and TOR

Directed cell migration and cell polarity are crucial in many facets of biological processes. Cellular motility requires a complex array of signaling pathways, in which orchestrated cross-talk, a feedback loop, and multi-component signaling recur. Almost every signaling molecule requires several regulatory processes to be functionally activated, and a lack of a signaling molecule often leads to chemotaxis defects, suggesting an integral role for each component in the pathway. We outline our current understanding of the signaling event that regulates chemotaxis with an emphasis on recent findings associated with the Ras, PI3K, and target of rapamycin (TOR) pathways and the interplay of these pathways. Ras, PI3K, and TOR are known as key regulators of cellular growth. Deregulation of those pathways is associated with many human diseases, such as cancer, developmental disorders, and immunological deficiency. Recent studies in yeast, mammalian cells, and Dictyostelium discoideum reveal another critical role of Ras, PI3K, and TOR in regulating the actin cytoskeleton, cell polarity, and cellular movement. These findings shed light on the mechanism by which eukaryotic cells maintain cell polarity and directed cell movement, and also demonstrate that multiple steps in the signal transduction pathway coordinately regulate cell motility.     

TOR信号调控真菌细胞的生长与代谢

TOR(target of rapamycin)是一类进化上保守的丝氨酸/苏氨酸(Ser/Thr)蛋白激酶,是真核细胞响应环境信号调控生长和代谢的关键因子。真菌TOR信号途径在营养、压力环境等刺激下,通过核糖体生物合成、营养物质摄入及代谢等过程调节维持胞内稳态。本文主要综述了酵母细胞TOR及TOR复合物的结构,以及近年来真菌TORC1蛋白在不同营养环境、压力等条件下对细胞生长与自噬、代谢以及胁迫生理响应等生命活动的调控机制进展及未来发展方向,为真菌TOR调控生长和代谢产物提供新思路。     

Insulin-induced changes in metabolism-related proteins during maize germination

Insulin regulates a wide range of metabolic processes in mammals, such as homeostasis and the breakdown of glucose. Recently, the existence of an insulin-related growth factor in maize (ZmIGF) and a possible receptor for this growth factor has been reported. This peptide exerts effects on plant growth and promotes germination by activating the target of rapamycin (TOR) signaling pathways, which is similar to the insulin response in mammals. In this study, we analyzed the insulin response in maize embryos using a proteomic approach. Our results indicated that insulin modulates the expression of proteins involved in processes, such as storage protein degradation, protein processing, redox and desiccation stress, and glucose metabolism. The involvement of TOR signaling pathways was analyzed using the TOR inhibitor, rapamycin. The results showed that the modulation of these proteins by insulin is independent of the TOR pathway. These results indicated that insulin promotes changes in metabolism-related proteins to ensure successful germination in maize.     

TOR1 and TOR2 are structurally and functionally similar but not identical phosphatidylinositol kinase homologues in yeast.

The Saccharomyces cerevisiae genes TOR1 and TOR2 were originally identified by mutations that confer resistance to the immunosuppressant rapamycin. TOR2 was previously shown to encode an essential 282-kDa phosphatidylinositol kinase (PI kinase) homologue. The TOR1 gene product is also a large (281 kDa) PI kinase homologue, with 67% identity to TOR2. TOR1 is not essential, but a TOR1 TOR2 double disruption uniquely confers a cell cycle (G1) arrest as does exposure to rapamycin; disruption of TOR2 alone is lethal but does not cause a cell cycle arrest. TOR1-TOR2 and TOR2-TOR1 hybrids indicate that carboxy-terminal domains of TOR1 and TOR2 containing a lipid kinase sequence motif are interchangeable and therefore functionally equivalent; the other portions of TOR1 and TOR2 are not interchangeable. The TOR1-1 and TOR2-1 mutations, which confer rapamycin resistance, alter the same potential protein kinase C site in the respective protein's lipid kinase domain. Thus, TOR1 and TOR2 are likely similar but not identical, rapamycin-sensitive PI kinases possibly regulated by phosphorylation. TOR1 and TOR2 may be components of a novel signal transduction pathway controlling progression through G1.     

Synergism between altered cortical polarity and the PI3K/TOR pathway in the suppression of tumour growth

Loss of function of pins (partner of inscuteable) partially disrupts neuroblast (NB) polarity and asymmetric division, results in fewer and smaller NBs and inhibits Drosophila larval brain growth. Food deprivation also inhibits growth. However, we find that the combination of loss of function of pins and dietary restriction results in loss of NB asymmetry, overproliferation of Miranda-expressing cells, brain overgrowth and increased frequency of tumour growth on allograft transplantation. The same effects are observed in well-fed pins larvae that are mutant for pi3k (phosphatidylinositol 3-kinase) or exposed to the TOR inhibitor rapamycin. Thus, pathways that are sensitive to food deprivation and dependent on PI3K and TOR are essential to suppress tumour growth in Drosophila larval brains with compromised pins function. These results highlight an unexpected crosstalk whereby the normally growth-promoting, nutrient-sensing PI3K/TOR pathway suppresses tumour formation in neural stem cells with compromised cell polarity.     

<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> FKBP12 binds <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> TOR and its expression in plants leads to rapamycin susceptibility

Background  

The eukaryotic TOR pathway controls translation, growth and the cell cycle in response to environmental signals such as nutrients or growth-stimulating factors. The TOR protein kinase can be inactivated by the antibiotic rapamycin following the formation of a ternary complex between TOR, rapamycin and FKBP12 proteins. The TOR protein is also found in higher plants despite the fact that they are rapamycin insensitive. Previous findings using the yeast two hybrid system suggest that the FKBP12 plant homolog is unable to form a complex with rapamycin and TOR, while the FRB domain of plant TOR is still able to bind to heterologous FKBP12 in the presence of rapamycin. The resistance to rapamycin is therefore limiting the molecular dissection of the TOR pathway in higher plants.     

Zebrafish Offers New Perspective on Developmental Role of TOR Signaling

Genetic studies on the molecular basis of growth control have converged on the target of rapamycin (TOR) pathway as a key regulator. When stimulated by nutrients (i.e. amino acids) or growth factors (i.e. insulin), TOR activates protein synthesis and other anabolic pathways to promote cell growth. Our knowledge of TOR's function in vivo is still rudimentary, particularly in the setting of vertebrate development. An important question is whether TOR functions as a constitutive regulator of growth in all cell types, or as a stage- and organ-specific regulator. Recently we employed the zebrafish as a vertebrate model system to study the developmental role of TOR signaling. We found that TOR signaling was required for a discrete step prior to epithelial differentiation. The results support the view that different organs may be reliant on TOR activity to differing degrees. In the case of the zebrafish, the digestive tract exhibits the greatest sensitivity to rapamycin, which may reflect its reliance on TOR signaling for normal growth. We suggest the hypothesis that TOR signaling may regulate the size of the intestine's absorptive surface area in response to systemic nutrient demand.     

SREBP activity is regulated by mTORC1 and contributes to Akt-dependent cell growth

Cell growth (accumulation of mass) needs to be coordinated with metabolic processes that are required for the synthesis of macromolecules. The PI3-kinase/Akt signaling pathway induces cell growth via activation of complex 1 of the target of rapamycin (TORC1). Here we show that Akt-dependent lipogenesis requires mTORC1 activity. Furthermore, nuclear accumulation of the mature form of the sterol responsive element binding protein (SREBP1) and expression of SREBP target genes was blocked by the mTORC1 inhibitor rapamycin. We also show that silencing of SREBP blocks Akt-dependent lipogenesis and attenuates the increase in cell size in response to Akt activation in vitro. Silencing of dSREBP in flies caused a reduction in cell and organ size and blocked the induction of cell growth by dPI3K. Our results suggest that the PI3K/Akt/TOR pathway regulates protein and lipid biosynthesis in an orchestrated manner and that both processes are required for cell growth.     

Rheb promotes cell growth as a component of the insulin/TOR signalling network

Insulin signalling is a potent stimulator of cell growth and has been proposed to function, at least in part, through the conserved protein kinase TOR (target of rapamycin) [corrected]. Recent studies suggest that the tuberous sclerosis complex Tsc1-Tsc2 may couple insulin signalling to Tor activity [corrected]. However, the regulatory mechanism involved remains unclear, and additional components are most probably involved. In a screen for novel regulators of growth, we identified Rheb (Ras homologue enriched in brain), a member of the Ras superfamily of GTP-binding proteins. Increased levels of Rheb in Drosophila melanogaster promote cell growth and alter cell cycle kinetics in multiple tissues. In mitotic tissues, overexpression of Rheb accelerates passage through G1-S phase without affecting rates of cell division, whereas in endoreplicating tissues, Rheb increases DNA ploidy. Mutation of Rheb suspends larval growth and prevents progression from first to second instar. Genetic and biochemical tests indicate that Rheb functions in the insulin signalling pathway downstream of Tsc1-Tsc2 and upstream of TOR. Levels of rheb mRNA are rapidly induced in response to protein starvation, and overexpressed Rheb can drive cell growth in starved animals, suggesting a role for Rheb in the nutritional control of cell growth.     

p70 S6 kinase-mediated protein synthesis is a critical step for vascular endothelial cell proliferation.

In this work, we analyzed the role of the PI3K-p70 S6 kinase (S6K) signaling cascade in the stimulation of endothelial cell proliferation. We found that inhibitors of the p42/p44 MAPK pathway (PD98059) and the PI3K-p70 S6K pathway (wortmannin, Ly294002, and rapamycin) all block thymidine incorporation stimulated by fetal calf serum in the resting mouse endothelial cell line 1G11. The action of rapamycin can be generalized, since it completely inhibits the mitogenic effect of fetal calf serum in primary endothelial cell cultures (human umbilical vein endothelial cells) and another established capillary endothelial cell line (LIBE cells). The inhibitory effect of rapamycin is only observed when the inhibitor is added at the early stages of G(0)-G(1) progression, suggesting an inhibitory action early in G(1). Rapamycin completely inhibits growth factor stimulation of protein synthesis, which perfectly correlates with the inhibition of cell proliferation. In accordance with its inhibitory action on protein synthesis, activation of cyclin D1 and p21 proteins by growth factors is also blocked by preincubation with rapamycin. Expression of a p70 S6K mutant partially resistant to rapamycin reverses the inhibitory effect of the drug on DNA synthesis, indicating that rapamycin action is via p70 S6K. Thus, in vascular endothelial cells, activation of protein synthesis via p70 S6K is an essential step for cell cycle progression in response to growth factors.     

Two TOR complexes,only one of which is rapamycin sensitive,have distinct roles in cell growth control

The target of rapamycin (TOR) proteins in Saccharomyces cerevisiae, TOR1 and TOR2, redundantly regulate growth in a rapamycin-sensitive manner. TOR2 additionally regulates polarization of the actin cytoskeleton in a rapamycin-insensitive manner. We describe two functionally distinct TOR complexes. TOR Complex 1 (TORC1) contains TOR1 or TOR2, KOG1 (YHR186c), and LST8. TORC2 contains TOR2, AVO1 (YOL078w), AVO2 (YMR068w), AVO3 (YER093c), and LST8. FKBP-rapamycin binds TORC1, and TORC1 disruption mimics rapamycin treatment, suggesting that TORC1 mediates the rapamycin-sensitive, TOR-shared pathway. FKBP-rapamycin fails to bind TORC2, and TORC2 disruption causes an actin defect, suggesting that TORC2 mediates the rapamycin-insensitive, TOR2-unique pathway. Thus, the distinct TOR complexes account for the diversity, specificity, and selective rapamycin inhibition of TOR signaling. TORC1 and possibly TORC2 are conserved from yeast to man.     

FKBP12-rapamycin target TOR2 is a vacuolar protein with an associated phosphatidylinositol-4 kinase activity.

In complex with the immunophilin FKBP12, the natural product rapamycin inhibits signal transduction events required for G1 to S phase cell cycle progression in yeast and mammalian cells. Genetic studies in yeast first implicated the TOR1 and TOR2 proteins as targets of the FKBP12-rapamycin complex. We report here that the TOR2 protein is membrane associated and localized to the surface of the yeast vacuole. Immunoprecipitated TOR2 protein contains readily detectable phosphatidylinositol-4 (PI-4) kinase activity attributable to either a TOR2 intrinsic activity or to a PI-4 kinase tightly associated with TOR2. Importantly, we find that rapamycin stimulates FKBP12 binding to wild-type TOR2 but not to a rapamycin-resistant TOR2-1 mutant protein. Surprisingly, FKBP12-rapamycin binding does not markedly inhibit the PI kinase activity associated with TOR2, but does cause a delocalization of TOR2 from the vacuolar surface, which may deprive the TOR2-associated PI-4 kinase activity of its in vivo substrate. Several additional findings indicate that vacuolar localization is important for TOR2 function and, conversely, that TOR2 modulates vacuolar morphology and segregation. These studies demonstrate that TOR2 is an essential, highly conserved component of a signal transduction pathway regulating cell cycle progression conserved from yeast to man.     

Regulation of the cell integrity pathway by rapamycin-sensitive TOR function in budding yeast

The TOR (target of rapamycin) pathway controls cell growth in response to nutrient availability in eukaryotic cells. Inactivation of TOR function by rapamycin or nutrient exhaustion is accompanied by triggering various cellular mechanisms aimed at overcoming the nutrient stress. Here we report that in Saccharomyces cerevisiae the protein kinase C (PKC)-mediated mitogen-activated protein kinase pathway is regulated by TOR function because upon specific Tor1 and Tor2 inhibition by rapamycin, Mpk1 is activated rapidly in a process mediated by Sit4 and Tap42. Osmotic stabilization of the plasma membrane prevents both Mpk1 activation by rapamycin and the growth defect that occurs upon the simultaneous absence of Tor1 and Mpk1 function, suggesting that, at least partially, TOR inhibition is sensed by the PKC pathway at the cell envelope. This process involves activation of cell surface sensors, Rom2, and downstream elements of the mitogen-activated protein kinase cascade. Rapamycin also induces depolarization of the actin cytoskeleton through the TOR proteins, Sit4 and Tap42, in an osmotically suppressible manner. Finally, we show that entry into stationary phase, a physiological situation of nutrient depletion, also leads to the activation of the PKC pathway, and we provide further evidence demonstrating that Mpk1 is essential for viability once cells enter G(0).