mTORC2 Responds to Glutamine Catabolite Levels to Modulate the Hexosamine Biosynthesis Enzyme GFAT1.

Highly proliferating cells are particularly dependent on glucose and glutamine for bioenergetics and macromolecule biosynthesis. The signals that respond to nutrient fluctuations to maintain metabolic homeostasis remain poorly understood. Here, we found that mTORC2 is activated by nutrient deprivation due to decreasing glutamine catabolites. We elucidate how mTORC2 modulates a glutamine-requiring biosynthetic pathway, the hexosamine biosynthesis pathway (HBP) via regulation of expression of glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1), the rate-limiting enzyme of the HBP. GFAT1 expression is dependent on sufficient amounts of glutaminolysis catabolites particularly α-ketoglutarate, which are generated in an mTORC2-dependent manner. Additionally, mTORC2 is essential for proper expression and nuclear accumulation of the GFAT1 transcriptional regulator, Xbp1s. Thus, while mTORC1 senses amino acid abundance to promote anabolism, mTORC2 responds to declining glutamine catabolites in order to restore metabolic homeostasis. Our findings uncover the role of mTORC2 in metabolic reprogramming and have implications for understanding insulin resistance and tumorigenesis.

The RNA-binding protein AUF1 facilitates Akt phosphorylation at the membrane.

Mammalian target of rapamycin (mTOR), which is part of mTOR complex 1 (mTORC1) and mTORC2, controls cellular metabolism in response to levels of nutrients and other growth signals. A hallmark of mTORC2 activation is the phosphorylation of Akt, which becomes upregulated in cancer. How mTORC2 modulates Akt phosphorylation remains poorly understood. Here, we found that the RNA-binding protein, AUF1 (ARE/poly(U)-binding/degradation factor 1), modulates mTORC2/Akt signaling. We determined that AUF1 is required for phosphorylation of Akt at Thr308, Thr450, and Ser473 and that AUF1 also mediates phosphorylation of the mTORC2-modulated metabolic enzyme glutamine fructose-6-phosphate amidotransferase 1 at Ser243. In addition, AUF1 immunoprecipitation followed by quantitative RT-PCR revealed that the mRNAs of Akt, glutamine fructose-6-phosphate amidotransferase 1, and the mTORC2 component SIN1 associate with AUF1. Furthermore, expression of the p40 and p45, but not the p37 or p42, isoforms of AUF1 specifically mediate Akt phosphorylation. In the absence of AUF1, subcellular fractionation indicated that Akt fails to localize to the membrane. However, ectopic expression of a membrane-targeted allele of Akt is sufficient to allow Akt-Ser473 phosphorylation despite AUF1 depletion. Finally, conditions that enhance mTORC2 signaling, such as acute glutamine withdrawal, augment AUF1 phosphorylation, whereas mTOR inhibition abolishes AUF1 phosphorylation. Our findings unravel a role for AUF1 in promoting membrane localization of Akt to facilitate its phosphorylation on this cellular compartment. Targeting AUF1 could have therapeutic benefit for cancers with upregulated mTORC2/Akt signaling.

mTORC2 modulates the amplitude and duration of GFAT1 Ser-243 phosphorylation to maintain flux through the hexosamine pathway during starvation.

The mechanistic target of rapamycin (mTOR) controls metabolic pathways in response to nutrients. Recently, we have shown that mTOR complex 2 (mTORC2) modulates the hexosamine biosynthetic pathway (HBP) by promoting the expression of the key enzyme of the HBP, glutamine:fructose-6-phosphate aminotransferase 1 (GFAT1). Here, we found that GFAT1 Ser-243 phosphorylation is also modulated in an mTORC2-dependent manner. In response to glutamine limitation, active mTORC2 prolongs the duration of Ser-243 phosphorylation, albeit at lower amplitude. Blocking glycolysis using 2-deoxyglucose robustly enhances Ser-243 phosphorylation, correlating with heightened mTORC2 activation, increased AMPK activity, and O-GlcNAcylation. However, when 2-deoxyglucose is combined with glutamine deprivation, GFAT1 Ser-243 phosphorylation and mTORC2 activation remain elevated, whereas AMPK activation and O-GlcNAcylation diminish. Phosphorylation at Ser-243 promotes GFAT1 expression and production of GFAT1-generated metabolites including ample production of the HBP end-product, UDP-GlcNAc, despite nutrient starvation. Hence, we propose that the mTORC2-mediated increase in GFAT1 Ser-243 phosphorylation promotes flux through the HBP to maintain production of UDP-GlcNAc when nutrients are limiting. Our findings provide insights on how the HBP is reprogrammed via mTORC2 in nutrient-addicted cancer cells.

Mammalian target of rapamycin complex 2 modulates αßTCR processing and surface expression during thymocyte development.

An efficient immune response relies on the presence of T cells expressing a functional TCR. Whereas the mechanisms generating TCR diversity for antigenic recognition are well defined, what controls its surface expression is less known. In this study, we found that deletion of the mammalian target of rapamycin complex (mTORC) 2 component rictor at early stages of T cell development led to aberrant maturation and increased proteasomal degradation of nascent TCRs. Although CD127 expression became elevated, the levels of TCRs as well as CD4, CD8, CD69, Notch, and CD147 were significantly attenuated on the surface of rictor-deficient thymocytes. Diminished expression of these receptors led to suboptimal signaling, partial CD4(-)CD8(-) double-negative 4 (CD25(-)CD44(-)) proliferation, and CD4(+)CD8(+) double-positive activation as well as developmental blocks at the CD4(-)CD8(-) double-negative 3 (CD25(+)CD44(-)) and CD8-immature CD8(+) single-positive stages. Because CD147 glycosylation was also defective in SIN1-deficient fibroblasts, our findings suggest that mTORC2 is involved in the co/posttranslational processing of membrane receptors. Thus, mTORC2 impacts development via regulation of the quantity and quality of receptors important for cell differentiation.

Inhibition of p70 Ribosomal S6 Kinase (S6K1) Reduces Cortical Blood Flow in a Rat Model of Autism-Tuberous Sclerosis.

The manifestations of tuberous sclerosis complex (TSC) in humans include epilepsy, autism spectrum disorders (ASD) and intellectual disability. Previous studies suggested the linkage of TSC to altered cerebral blood flow and metabolic dysfunction. We previously reported a significant elevation in cerebral blood flow in an animal model of TSC and autism of young Eker rats. Inhibition of the mammalian target of rapamycin (mTOR) by rapamycin could restore normal oxygen consumption and cerebral blood flow. In this study, we investigated whether inhibiting a component of the mTOR signaling pathway, p70 ribosomal S6 kinase (S6K1), would yield comparable effects. Control Long Evans and Eker rats were divided into vehicle and PF-4708671 (S6K1 inhibitor, 75 mg/kg for 1 h) treated groups. Cerebral regional blood flow (14C-iodoantipyrine) was determined in isoflurane anesthetized rats. We found significantly increased basal cortical (+ 32%) and hippocampal (+ 15%) blood flow in the Eker rats. PF-4708671 significantly lowered regional blood flow in the cortex and hippocampus of the Eker rats. PF-4708671 did not significantly lower blood flow in these regions in the control Long Evans rats. Phosphorylation of S6-Ser240/244 and Akt-Ser473 was moderately decreased in Eker rats but only the latter reached statistical significance upon PF-4708671 treatment. Our findings suggest that moderate inhibition of S6K1 with PF-4708671 helps to restore normal cortical blood flow in Eker rats and that this information might have therapeutic potential in tuberous sclerosis complex and autism.

Fascin1 promotes cell migration of mature dendritic cells.

Dendritic cells (DCs) play central roles in innate and adaptive immunity. Upon maturation, DCs assemble numerous veil-like membrane protrusions, disassemble podosomes, and travel from the peripheral tissues to lymph nodes to present Ags to T cells. These alterations in morphology and motility are closely linked to the primary function of DCs, Ag presentation. However, it is unclear how and what cytoskeletal proteins control maturation-associated alterations, in particular, the change in cell migration. Fascin1, an actin-bundling protein, is specifically and greatly induced upon maturation, suggesting a unique role for fascin1 in mature DCs. To determine the physiological roles of fascin1, we characterized bone marrow-derived, mature DCs from fascin1 knockout mice. We found that fascin1 is critical for cell migration: fascin1-null DCs exhibit severely decreased membrane protrusive activity. Importantly, fascin1-null DCs have lower chemotactic activity toward CCL19 (a chemokine for mature DCs) in vitro, and in vivo, Langerhans cells show reduced emigration into draining lymph nodes. Morphologically, fascin1-null mature DCs are flatter and fail to disassemble podosomes, a specialized structure for cell-matrix adhesion. Expression of exogenous fascin1 in fascin1-null DCs rescues the defects in membrane protrusive activity, as well as in podosome disassembly. These results indicate that fascin1 positively regulates migration of mature DCs into lymph nodes, most likely by increasing dynamics of membrane protrusions, as well as by disassembling podosomes.

The Hexosamine Biosynthesis Pathway: Regulation and Function.

The hexosamine biosynthesis pathway (HBP) produces uridine diphosphate-N-acetyl glucosamine, UDP-GlcNAc, which is a key metabolite that is used for N- or O-linked glycosylation, a co- or post-translational modification, respectively, that modulates protein activity and expression. The production of hexosamines can occur via de novo or salvage mechanisms that are catalyzed by metabolic enzymes. Nutrients including glutamine, glucose, acetyl-CoA, and UTP are utilized by the HBP. Together with availability of these nutrients, signaling molecules that respond to environmental signals, such as mTOR, AMPK, and stress-regulated transcription factors, modulate the HBP. This review discusses the regulation of GFAT, the key enzyme of the de novo HBP, as well as other metabolic enzymes that catalyze the reactions to produce UDP-GlcNAc. We also examine the contribution of the salvage mechanisms in the HBP and how dietary supplementation of the salvage metabolites glucosamine and N-acetylglucosamine could reprogram metabolism and have therapeutic potential. We elaborate on how UDP-GlcNAc is utilized for N-glycosylation of membrane and secretory proteins and how the HBP is reprogrammed during nutrient fluctuations to maintain proteostasis. We also consider how O-GlcNAcylation is coupled to nutrient availability and how this modification modulates cell signaling. We summarize how deregulation of protein N-glycosylation and O-GlcNAcylation can lead to diseases including cancer, diabetes, immunodeficiencies, and congenital disorders of glycosylation. We review the current pharmacological strategies to inhibit GFAT and other enzymes involved in the HBP or glycosylation and how engineered prodrugs could have better therapeutic efficacy for the treatment of diseases related to HBP deregulation.

Thymic selection threshold defined by compartmentalization of Ras/MAPK signalling.

A healthy individual can mount an immune response to exogenous pathogens while avoiding an autoimmune attack on normal tissues. The ability to distinguish between self and non-self is called 'immunological tolerance' and, for T lymphocytes, involves the generation of a diverse pool of functional T cells through positive selection and the removal of overtly self-reactive thymocytes by negative selection during T-cell ontogeny. To elucidate how thymocytes arrive at these cell fate decisions, here we have identified ligands that define an extremely narrow gap spanning the threshold that distinguishes positive from negative selection. We show that, at the selection threshold, a small increase in ligand affinity for the T-cell antigen receptor leads to a marked change in the activation and subcellular localization of Ras and mitogen-activated protein kinase (MAPK) signalling intermediates and the induction of negative selection. The ability to compartmentalize signalling molecules differentially in the cell endows the thymocyte with the ability to convert a small change in analogue input (affinity) into a digital output (positive versus negative selection) and provides the basis for establishing central tolerance.

Dietary glucosamine overcomes the defects in αß-T cell ontogeny caused by the loss of de novo hexosamine biosynthesis.

T cell development requires the coordinated rearrangement of T cell receptor (TCR) gene segments and the expression of either αß or γδ TCR. However, whether and how de novo synthesis of nutrients contributes to thymocyte commitment to either lineage remains unclear. Here, we find that T cell-specific deficiency in glutamine:fructose-6-phosphate aminotransferase 1 (GFAT1), the rate-limiting enzyme of the de novo hexosamine biosynthesis pathway (dn-HBP), attenuates hexosamine levels, blunts N-glycosylation of TCRß chains, reduces surface expression of key developmental receptors, thus impairing αß-T cell ontogeny. GFAT1 deficiency triggers defects in N-glycans, increases the unfolded protein response, and elevates  γδ-T cell numbers despite reducing γδ-TCR diversity. Enhancing TCR expression or PI3K/Akt signaling does not reverse developmental defects. Instead, dietary supplementation with the salvage metabolite, glucosamine, and an α-ketoglutarate analogue partially restores αß-T cell development in GFAT1T-/- mice, while fully rescuing it in ex vivo fetal thymic organ cultures. Thus, dn-HBP fulfils, while salvage nutrients partially satisfy, the elevated demand for hexosamines during early T cell development.

The death effector domain protein PEA-15 negatively regulates T-cell receptor signaling.

PEA-15 is a death effector domain-containing phosphoprotein that binds ERK and restricts it to the cytoplasm. PEA-15 also binds to FADD and thereby blocks apoptosis induced by death receptors. Abnormal expression of PEA-15 is associated with type II diabetes and some cancers; however, its physiological function remains unclear. To determine the function of PEA-15 in vivo, we used C57BL/6 mice in which the PEA-15 coding region was deleted. We thereby found that PEA-15 regulates T-cell proliferation. PEA-15-null mice did not have altered thymic or splenic lymphocyte cellularity or differentiation. However, PEA-15 deficient T cells had increased CD3/CD28-induced nuclear translocation of ERK and increased activation of IL-2 transcription and secretion in comparison to control wild-type littermates. Indeed, activation of the T-cell receptor in wild-type mice caused PEA-15 release of ERK. In contrast, overexpression of PEA-15 in Jurkat T cells blocked nuclear translocation of ERK and IL-2 transcription. Finally, PEA-15-null T cells showed increased IL-2 dependent proliferation on stimulation. No differences in T cell susceptibility to apoptosis were found. Thus, PEA-15 is a novel player in T-cell homeostasis. As such this work may have far reaching implications in understanding how the immune response is controlled.

MTOR Signaling and Metabolism in Early T Cell Development.

The mechanistic target of rapamycin (mTOR) controls cell fate and responses via its functions in regulating metabolism. Its role in controlling immunity was unraveled by early studies on the immunosuppressive properties of rapamycin. Recent studies have provided insights on how metabolic reprogramming and mTOR signaling impact peripheral T cell activation and fate. The contribution of mTOR and metabolism during early T-cell development in the thymus is also emerging and is the subject of this review. Two major T lineages with distinct immune functions and peripheral homing organs diverge during early thymic development; the αß- and γδ-T cells, which are defined by their respective TCR subunits. Thymic T-regulatory cells, which have immunosuppressive functions, also develop in the thymus from positively selected αß-T cells. Here, we review recent findings on how the two mTOR protein complexes, mTORC1 and mTORC2, and the signaling molecules involved in the mTOR pathway are involved in thymocyte differentiation. We discuss emerging views on how metabolic remodeling impacts early T cell development and how this can be mediated via mTOR signaling.

A new function for LAT and CD8 during CD8-mediated apoptosis that is independent of TCR signal transduction.

The majority (>95%) of thymocytes undergo apoptosis during selection in the thymus. Several mechanisms have been proposed to explain how apoptosis of thymocytes that are not positively selected occurs; however, it is unknown whether thymocytes die purely by "neglect" or whether signaling through a cell-surface receptor initiates an apoptotic pathway. We have previously demonstrated that on double positive thymocytes the ligation of CD8 in the absence of TCR engagement results in apoptosis and have postulated this is a mechanism to remove thymocytes that have failed positive selection. On mature single positive T cells CD8 acts as a co-receptor to augment signaling through the TCR that is dependent on the phosphorylation of the adaptor protein, linker for activation of T cells (LAT). Here, we show that during CD8-mediated apoptosis of double positive thymocytes there is an increase in the association of CD8 with LAT and an increase in LAT tyrosine phosphorylation. Decreasing LAT expression and mutation of tyrosine residues of LAT reduced apoptosis upon crosslinking of CD8. Our results identify novel functions for both CD8 and LAT that are independent of TCR signal transduction and suggest a mechanism for signal transduction leading to apoptosis upon CD8 crosslinking.

mTORC2 Is Involved in the Induction of RSK Phosphorylation by Serum or Nutrient Starvation.

Cells adjust to nutrient fluctuations to restore metabolic homeostasis. The mechanistic target of rapamycin (mTOR) complex 2 responds to nutrient levels and growth signals to phosphorylate protein kinases belonging to the AGC (Protein Kinases A,G,C) family such as Akt and PKC. Phosphorylation of these AGC kinases at their conserved hydrophobic motif (HM) site by mTORC2 enhances their activation and mediates the functions of mTORC2 in cell growth and metabolism. Another AGC kinase family member that is known to undergo increased phosphorylation at the homologous HM site (Ser380) is the p90 ribosomal S6 kinase (RSK). Phosphorylation at Ser380 is facilitated by the activation of the mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) in response to growth factor stimulation. Here, we demonstrate that optimal phosphorylation of RSK at this site requires an intact mTORC2. We also found that RSK is robustly phosphorylated at Ser380 upon nutrient withdrawal or inhibition of glycolysis, conditions that increase mTORC2 activation. However, pharmacological inhibition of mTOR did not abolish RSK phosphorylation at Ser380, indicating that mTOR catalytic activity is not required for this phosphorylation. Since RSK and SIN1ß colocalize at the membrane during serum restimulation and acute glutamine withdrawal, mTORC2 could act as a scaffold to enhance RSK HM site phosphorylation. Among the known RSK substrates, the CCTß subunit of the chaperonin containing TCP-1 (CCT) complex had defective phosphorylation in the absence of mTORC2. Our findings indicate that the mTORC2-mediated phosphorylation of the RSK HM site could confer RSK substrate specificity and reveal that RSK responds to nutrient fluctuations.

Lck regulates the threshold of activation in primary T cells, while both Lck and Fyn contribute to the magnitude of the extracellular signal-related kinase response.

The src family kinases p56lck (Lck) and p59fyn (Fyn) are the most proximal signaling molecules to be activated downstream of the T-cell receptor. Using an inducible transgenic model, we can regulate the expression of Lck in primary T cells and ask how the signaling cascade and differentiation potential are affected by the absence or the presence of reduced levels of Lck. We show that in naïve T cells, Lck controls the threshold of activation by preferentially regulating multiple signaling pathways that result in the mobilization of Ca2+ through activation of phospholipase C-gamma and protein kinase C as well as activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway. Fyn is also able to stimulate the ERK/MAPK pathway in primary T cells but has little influence on the mobilization of Ca2+. Only Lck efficiently stimulates production of diacylglycerol and therefore RasGRP1 recruitment to the plasma membrane and phosphorylation of Shc, suggesting that Fyn activates ERK via a different upstream signaling route. Finally, we show that signals through Lck are essential for the development of T-cell-effector potential, particularly for effective cytokine transcription.

The T-cell receptor signalosome: a dynamic structure with expanding complexity.

Signal transduction in T cells is a dynamic process involving a large number of membrane and cytosolic proteins. The TCR macromolecular complex (signalosome) is initiated by receptor occupancy and becomes more elaborate over time. This review describes how 'vertical' displacement mechanisms and lateral coalescence of lipid-raft-associated scaffold proteins combine to form distinct signalosomes, which control signal specificity.

Signaling life and death in the thymus: timing is everything.

T lymphocytes are generated in the thymus, where developing thymocytes must accept one of two fates: They either differentiate or they die. These fates are chiefly determined by signals that originate from the T cell receptor (TCR), a single receptor complex with a remarkable capacity to decide between distinct cell fates. This review explores TCR signaling in thymocytes and focuses on the kinetic aspects of ligand binding, coreceptor involvement, protein phosphorylation, and mitogen-activated protein kinase (MAPK) activation. Understanding the logic of TCR signaling may eventually explain how thymocytes and T cells distinguish self from nonself, a phenomenon that has fascinated immunologists for 50 years.