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.     

The PP2A regulatory subunit Tap46, a component of the TOR signaling pathway, modulates growth and metabolism in plants

Tap42/α4, a regulatory subunit of protein phosphatase 2A, is a downstream effector of the target of rapamycin (TOR) protein kinase, which regulates cell growth in coordination with nutrient and environmental conditions in yeast and mammals. In this study, we characterized the functions and phosphatase regulation of plant Tap46. Depletion of Tap46 resulted in growth arrest and acute plant death with morphological markers of programmed cell death. Tap46 interacted with PP2A and PP2A-like phosphatases PP4 and PP6. Tap46 silencing modulated cellular PP2A activities in a time-dependent fashion similar to TOR silencing. Immunoprecipitated full-length and deletion forms of Arabidopsis thaliana TOR phosphorylated recombinant Tap46 protein in vitro, supporting a functional link between Tap46 and TOR. Tap46 depletion reproduced the signature phenotypes of TOR inactivation, such as dramatic repression of global translation and activation of autophagy and nitrogen mobilization, indicating that Tap46 may act as a positive effector of TOR signaling in controlling those processes. Additionally, Tap46 silencing in tobacco (Nicotiana tabacum) BY-2 cells caused chromatin bridge formation at anaphase, indicating its role in sister chromatid segregation. These findings suggest that Tap46, in conjunction with associated phosphatases, plays an essential role in plant growth and development as a component of the TOR signaling pathway.     

The TOR nutrient signalling pathway phosphorylates NPR1 and inhibits turnover of the tryptophan permease.

The Saccharomyces cerevisiae targets of rapamycin, TOR1 and TOR2, signal activation of cell growth in response to nutrient availability. Loss of TOR or rapamycin treatment causes yeast cells to arrest growth in early G1 and to express several other physiological properties of starved (G0) cells. As part of this starvation response, high affinity amino acid permeases such as the tryptophan permease TAT2 are targeted to the vacuole and degraded. Here we show that the TOR signalling pathway phosphorylates the Ser/Thr kinase NPR1 and thereby inhibits the starvation-induced turnover of TAT2. Overexpression of NPR1 inhibits growth and induces the degradation of TAT2, whereas loss of NPR1 confers resistance to rapamycin and to FK506, an inhibitor of amino acid import. NPR1 is controlled by TOR and the type 2A phosphatase-associated protein TAP42. First, overexpression of NPR1 is toxic only when TOR function is reduced. Secondly, NPR1 is rapidly dephosphorylated in the absence of TOR. Thirdly, NPR1 dephosphorylation does not occur in a rapamycin-resistant tap42 mutant. Thus, the TOR nutrient signalling pathway also controls growth by inhibiting a stationary phase (G0) programme. The control of NPR1 by TOR is analogous to the control of p70 s6 kinase and 4E-BP1 by mTOR in mammalian cells.     

The TSC/Rheb/TOR Signaling Pathway in Fission Yeast and Mammalian Cells: Temperature Sensitive and Constitutive Active Mutants of TOR

The TSC/Rheb/TOR signaling pathway plays important roles in growth and cell cycle regulation. The main player TOR belongs to the PI3K-related protein kinase family. Recent studies utilizing fission yeast Tor2 have led to the identification of a number of amino acid changes that lead to inactivation as well as activation of TOR kinase. Also, constitutive active mutations in its upstream regulator, Rheb, have been identified. Isolation and characterization of temperature sensitive Tor2 mutants have established that this kinase functions as a key switch that determines cell fate between growth and sexual development. Introduction of Tor2 activating mutations into mTOR conferred nutrient independent activation of mTOR. Interestingly, these studies point to regions of TOR kinase important for its function.     

Molecular functions of the PP2A regulatory subunit Tap46 in plants

Tap42/α4 is a regulatory subunit of the protein phosphatase 2A (PP2A) family of phosphatases and plays a role in the target of rapamycin (TOR) pathway that regulates cell growth, ribosome biogenesis, translation and cell cycle progression in both yeast and mammals. We determined the cellular functions of Tap46, the plant homolog of Tap42/α4, in both Arabidopsis thaliana and Nicotiana benthamiana. Tap46 associated with the catalytic subunits of PP2A and the PP2A-like phosphatases PP4 and PP6 in vivo. Tap46 was phosphorylated by TOR in vitro, indicating that Tap46 is a direct substrate of TOR kinase. Tap46 deficiency caused cellular phenotypes that are similar to TOR-depletion phenotypes, including repression of global translation and activation of both autophagy and nitrogen recycling. Furthermore, Tap46 depletion regulated total PP2A activity in a time-dependent manner similar to TOR deficiency. These results suggest that Tap46 acts as a positive effector of the TOR signaling pathway in controlling diverse metabolic processes in plants. However, Tap46 silencing caused acute cell death, while TOR silencing only hastened senescence. Furthermore, mitotic cells with reduced Tap46 levels exhibited chromatin bridges at anaphase, while TOR depletion did not cause a similar defect. These findings suggest that Tap46 may have TOR-independent functions as well as functions related to TOR signaling in plants.Key words: acute cell death, autophagy, chromatin bridge, nitrogen mobilization, protein phosphatases, target of rapamycin (TOR)Yeast type 2A phosphatase-associated protein 42 kDa (Tap42) is a regulatory subunit that directly associates with catalytic subunits of the protein phosphatase 2A (PP2A) family of protein phosphatases to make a heterodimer and regulates the activity and substrate specificity of the intact enzyme complex.¹ Functions of Tap42 as a component of the target of rapamycin (TOR) signaling pathway have been well characterized in yeast.^1–3 Tap42-regulated phosphatase activities play a major role in signal transduction mediated by TOR. Accumulating evidence suggest that TOR regulates phosphorylation of target proteins by restraining PP2A activity through Tap42 phosphorylation.^1–3 Rapamycin inhibits TOR activity and also influences Tap42-mediated phosphatase regulation in yeast.^3–5α4, the mammalian homolog of Tap42, also associates with the catalytic subunits of PP2A, PP4 and PP6 to make a heterodimer.⁶ Rapamycin inhibits mammalian TOR (mTOR) activity, but it is not clear whether rapamycin prevents the formation of the α4/PP2Ac complex or whether α4 stimulates or represses PP2Ac activity.^7–9 Interestingly, loss of Tap42 function in Drosophila does not affect TOR-regulated activities, including cell growth, metabolism and S6 kinase activity, but results in mitotic arrest caused by spindle anomalies and subsequent activation of c-Jun N-terminal kinase signaling and apoptosis.¹⁰ Similarly, α4 deletion in mice leads to the rapid onset of apoptosis in both proliferating and differentiated cells, while rapamycin itself does not severely affect adult cells.¹¹ Furthermore, while TOR depletion causes developmental arrest and organ degeneration at the L3 stage in Caenorhabditis elegans, loss of α4 does not reproduce TOR deficiency phenotypes, but mainly leads to a fertility defect.¹² Taken together, these results suggest that the yeast Tap42/TOR paradigm is not completely conserved in higher eukaryotes and that Tap42/α4 functions may not be exclusively dependent on the Tor signaling pathway.In this study, we investigated the in vivo functions and phosphatase regulation of Tap46, the plant Tap42/α4 homolog, in relation to TOR in Nicotiana benthamiana, Arabidopsis and tobacco BY2 cells. Tap46 was shown to interact with the catalytic subunits of PP2A, PP4 and PP6 in vivo. Recombinant Tap46 protein was phosphorylated by immunoprecipitated TOR kinase and its deletion forms in vitro. Dexamethasone-induced RNAi of Tap46 caused dramatic repression of global translation and activation of both autophagy and nitrogen mobilization in the early stages of gene silencing. These phenotypes mimic those of TOR inactivation or TOR deficiency in Arabidopsis, yeast and mammals, indicating that Tap46 is a critical mediator of the Tor pathway in the regulation of these metabolic processes in plants. However, these early phenotypes of Tap46-deficient plants were soon followed by an acute and rapid programmed cell-death (PCD), while TOR silencing only led to growth retardation and premature senescence in Arabidopsis and N. benthamiana, confirming results from a previous study.¹³ The PCD caused by Tap46 deficiency is consistent with the apoptosis induced by loss of Tap42/α4 function in both Drosophila and mice.^10,11 Thus Tap42/α4/Tap46 appears to have a strong anti-apoptotic activity in higher eukaryotes. The underlying mechanisms of PCD activation caused by Tap46 depletion remain to be revealed, but it is possible that the inappropriate modulation of phosphatase activity and aberrant protein phosphorylation led to stress signaling and PCD activation.Another interesting phenotype of Tap46 deficiency is the formation of chromatin bridges in anaphase during mitosis, suggesting a role for Tap46 in plant cell mitotic progression. However, there have been no reports of anaphase bridge formation in tor mutants of any organisms. In Drosophila, loss of Tap42 function causes spindle disorganization and pre-anaphase arrest prior to the onset of apoptosis.¹⁰ In addition, Drosophila mutants with a defective regulatory subunit of PP2A exhibit an increased number of lagging chromosomes and chromatin bridges in anaphase.^14,15 Tap46 likely regulates the functions of PP2A family phosphatases during mitosis by direct association with their catalytic subunits, thereby modulating both the activity and specificity of the enzyme. Accumulating evidence reveals dynamic functions of PP2A during mitosis in both yeast and mammals: PP2A regulates kinetochore function, sister chromatid cohesion, spindle bipolarity and progression to anaphase.^15–17 Counteracting the activity of protein kinases, PP4 has also been implicated in both centrosome maturation and function during mitosis.¹⁸ Based on immunolabeling results, Tap46 was visualized as distinct spots around chromatin and mitotic spindles during mitosis in tobacco BY2 cells (Lee HS and Pai HS, unpublished results). Further studies will address the interacting partners and dynamic relocation of Tap46 during the cell cycle.Our results in this study demonstrated that Tap46 plays an important regulatory role in plant growth and metabolism; a major part of its function appears related to TOR signaling. However, we consistently observed certain phenotypic differences between Tap46-silenced and TOR-silenced Arabidopsis and N. benthamiana plants: an acute and rapid PCD occurred upon Tap46 silencing but not upon TOR silencing, despite a similar degree of gene silencing. Furthermore, we did not observe anaphase bridge formation in mitotic root-tip cells of ethanol-induced TOR RNAi Arabidopsis plants, while chromatin bridges were repeatedly observed in Tap46-silenced tobacco BY2 and Arabidopsis root-tip cells. Although an ancient Tap42/TOR paradigm observed in yeast appears to be conserved in plants, new TOR-independent functions of Tap46 might have evolved, the abrogation of which can cause massive PCD activation and anaphase bridge formation. Tap46 is a major regulator of cellular PP2A activity in plant cells by interacting with multiple phosphatase partners. Unraveling the molecular networks of Tap46 activity and interactions is essential for understanding its TOR-dependent and -independent functions in plants.     

The Association of Tap42-Phosphatase Complexes with TORC1 — Another Level of Regulation in Tor Signaling

In the budding yeast Saccharomyces cerevisiae, rapamycin has been known to induce a rapid dephosphorylation of many downstream targets of Tor. The key components mediating this dephosphorylation process are the Tap42-associated phosphatases, which become active upon rapamycin treatment. However, the mechanism by which rapamycin rapidly activates phosphatases is unclear. A recent report has provided evidence demonstrating a physical association of the Tap42-phosphatase complexes with TORC1, which is sensitive to rapamycin treatment or nutrient starvation. This association adds another level of regulation in Tor signaling, and explains why rapamycin or nutrient availability is able to initiate a rapid and robust response in the cell.     

Protein phosphatase 2A on track for nutrient-induced signalling in yeast

Early studies identified two bona fide protein phosphatase 2A (PP2A)-encoding genes in Saccharomyces cerevisiae, designated PPH21 and PPH22. In addition, three PP2A-related phosphatases, encoded by PPH3, SIT4 and PPG1, have been identified. All share as much as 86% sequence similarity at the amino acid level. This review will focus primarily on Pph21 and Pph22, but some aspects of Sit4 regulation will also be discussed. Whereas a role for PP2A in yeast morphology and cell cycle has been readily recognized, uncovering its function in yeast signal transduction is a more recent breakthrough. Via their interaction with phosphorylated Tap42, PP2A and Sit4 play a pivotal role in target of rapamycin (TOR) signalling. PPH22 overexpression mimics overactive cAMP-PKA (protein kinase A) signalling and PP2A and Sit4 might represent ceramide signalling targets. The methylation of its catalytic subunit stabilizes the heterotrimeric form of PP2A and might counteract TOR signalling. We will show how these new elements could lead us to understand the role and regulation of PP2A in nutrient-induced signalling in baker's yeast.     

Phosphatase-associated protein MoTip41 interacts with the phosphatase MoPpe1 to mediate crosstalk between TOR and cell wall integrity signalling during infection by the rice blast fungus Magnaporthe oryzae

The type 2A (PP2A) and type 2A-like (PP4 and PP6) serine/threonine phosphatases participate in a variety of cellular processes, such as cell cycle progression, signal transduction and apoptosis. Previously, we reported that the PP6 catalytic subunit MoPpe1, which interacts with and is suppressed by type 2A associated protein of 42 kDa (MoTap42), an essential protein involved in the target of rapamycin (TOR) signalling pathway, has important roles in development, virulence and activation of the cell wall integrity (CWI) pathway in the rice blast fungus Magnaporthe oryzae. Here, we show that Tap42-interacting protein 41 (MoTip41) mediates crosstalk between the TOR and CWI signalling pathways; and participates in the TOR pathway via interaction with MoPpe1, but not MoTap42. The deletion of MoTIP41 resulted in disruption of CWI signalling, autophagy, vegetative growth, appressorium function and plant infection, as well as increased sensitivity to rapamycin. Further investigation revealed that MoTip41 modulates activation of the CWI pathway in response to infection by interfering with the interaction between MoTap42 and MoPpe1. These findings enhance our understanding of how crosstalk between TOR and CWI signalling modulates the development and pathogenicity of M. oryzae.     

The phosphatase subunit tap42 functions independently of target of rapamycin to regulate cell division and survival in Drosophila

The protein phosphatase 2A (PP2A) regulatory subunit Tap42 is essential for target of rapamycin (TOR)-mediated signaling in yeast, but its role in higher eukaryotes has not been established. Here we show that Tap42 does not contribute significantly to TOR signaling in Drosophila, as disruption of the Tap42 gene does not cause defects in cell growth, metabolism, or S6-kinase activity characteristic of TOR inactivation. In addition, Tap42 is not required for increased cell growth in response to activation of TOR signaling. Instead, we find that Tap42 mutations cause disorganization of spindle microtubules in larval neuroblasts, leading to a preanaphase mitotic arrest in these cells. Loss of Tap42 ultimately results in increased JNK signaling, caspase activation, and cell death. These phenotypes are associated with increased accumulation and nuclear localization of PP2A in Tap42 mutant cells. Our results demonstrate that the role of Tap42 in TOR signaling has not been conserved in higher eukaryotes, indicating fundamental differences in the mechanisms of TOR signaling between yeast and higher eukaryotes.     

Role of Cryptococcus neoformans Rho1 GTPases in the PKC1 Signaling Pathway in Response to Thermal Stress

To initiate and establish infection in mammals, the opportunistic fungal pathogen Cryptococcus neoformans must survive and thrive upon subjection to host temperature. Primary maintenance of cell integrity is controlled through the protein kinase C1 (PKC1) signaling pathway, which is regulated by a Rho1 GTPase in Saccharomyces cerevisiae. We identified three C. neoformans Rho GTPases, Rho1, Rho10, and Rho11, and have begun to elucidate their role in growth and activation of the PKC1 pathway in response to thermal stress. Western blot analysis revealed that heat shock of wild-type cells resulted in phosphorylation of Mpk1 mitogen-activated protein kinase (MAPK). Constitutive activation of Rho1 caused phosphorylation of Mpk1 independent of temperature, indicating its role in pathway regulation. A strain with a deletion of RHO10 also displayed this constitutive Mpk1 phosphorylation phenotype, while one with a deletion of RHO11 yielded phosphorylation similar to that of wild type. Surprisingly, like a rho10Δ strain, a strain with a deletion of both RHO10 and RHO11 displayed temperature sensitivity but mimicked wild-type phosphorylation, which suggests that Rho10 and Rho11 have coordinately regulated functions. Heat shock-induced Mpk1 phosphorylation also required the PKC1 pathway kinases Bck1 and Mkk2. However, Pkc1, thought to be the major regulatory kinase of the cell integrity pathway, was dispensable for this response. Together, our results argue that Rho proteins likely interact via downstream components of the PKC1 pathway or by alternative pathways to activate the cell integrity pathway in C. neoformans.     

TOR complex 1 includes a novel component, Tco89p (YPL180w), and cooperates with Ssd1p to maintain cellular integrity in Saccharomyces cerevisiae

The Tor1p and Tor2p kinases, targets of the therapeutically important antibiotic rapamycin, function as components of two distinct protein complexes in yeast, termed TOR complex 1 (TORC1) and TORC2. TORC1 is responsible for a wide range of rapamycin-sensitive cellular activities and contains, in addition to Tor1p or Tor2p, two highly conserved proteins, Lst8p and Kog1p. By identifying proteins that co-purify with Tor1p, Tor2p, Lst8p, and Kog1p, we have characterized a comprehensive set of protein-protein interactions that define further the composition of TORC1 as well as TORC2. In particular, we have identified Tco89p (YPL180w) and Bit61p (YJL058c) as novel components of TORC1 and TORC2, respectively. Deletion of TOR1 or TCO89 results in two specific and distinct phenotypes, (i) rapamycin-hypersensitivity and (ii) decreased cellular integrity, both of which correlate with the presence of SSD1-d, an allele of SSD1 previously associated with defects in cellular integrity. Furthermore, we link Ssd1p to Tap42p, a component of the TOR pathway that is believed to act uniquely downstream of TORC1. Together, these results define a novel connection between TORC1 and Ssd1p-mediated maintenance of cellular integrity.