The mammalian target of rapamycin (mTOR) governs cell growth and proliferation by mediating the mitogen- and nutrient-dependent signal transduction that regulates messenger RNA translation. We identified phosphatidic acid (PA) as a critical component of mTOR signaling. In our study, mitogenic stimulation of mammalian cells led to a phospholipase D-dependent accumulation of cellular PA, which was required for activation of mTOR downstream effectors. PA directly interacted with the domain in mTOR that is targeted by rapamycin, and this interaction was positively correlated with mTOR's ability to activate downstream effectors. The involvement of PA in mTOR signaling reveals an important function of this lipid in signal transduction and protein synthesis, as well as a direct link between mTOR and mitogens. Furthermore, these studies suggest a potential mechanism for the in vivo actions of the immunosuppressant rapamycin.

Summary The evolutionarily conserved target of rapamycin (TOR) signaling controls growth, metabolism and aging. In the first robust demonstration of pharmacologically-induced life extension in a mammal, longevity was extended in mice treated with rapamycin, an inhibitor of mechanistic TOR (mTOR). However, detrimental metabolic effects of rapamycin treatment were also reported, presenting a paradox of improved survival despite metabolic impairment. How rapamycin extended lifespan in mice with such paradoxical effects was unclear. Here we show that detrimental effects of rapamycin treatment were only observed during the early stages of treatment. As the treatment continued for 20 weeks, these effects were reversed or diminished; the mice had better metabolic profiles, increased oxygen consumption and ketogenesis, and markedly enhanced insulin sensitivity. Thus, prolonged rapamycin treatment led to beneficial metabolic alterations, consistent with life extension previously observed. Our findings provide a likely explanation of the “rapamycin paradox” and support the potential causal importance of these metabolic alterations in longevity.

Dishevelled (Dsh) is a cytoplasmic multidomain protein that is required for all known branches of the Wnt signalling pathway1–3. The Frizzled/planar cell polarity (Fz/PCP) signalling branch requires an asymmetric cortical localization of Dsh, but this process remains poorly understood. Using a genome-wide RNA interference (RNAi) screen in Drosophila melanogaster cells, we show that Dsh membrane localization is dependent on the Na+/H+ exchange activity of the plasma membrane exchanger Nhe2. Manipulating Nhe2 expression levels in the eye causes PCP defects, and Nhe2 interacts genetically with Fz. Our data show that the binding and surface recruitment of Dsh by Fz is pH- and charge-dependent. We identify a polybasic stretch within the Dsh DEP domain that binds to negatively charged phospholipids and appears to be mechanistically important. Dsh recruitment by Fz can be abolished by converting these basic amino-acid residues into acidic ones, as in the mutant, DshKR/E. In vivo, the DshKR/E(2×) mutant with two substituted residues fails to associate with the membrane during active PCP signalling but rescues canonical Wnt signalling defects in a dsh-background. These results suggest that direct interaction between Fz and Dsh is stabilized by a pH and charge-dependent interaction of the DEP domain with phospholipids. This stabilization is particularly important for the PCP signalling branch and, thus, promotes specific pathway selection in Wnt signalling.

The mammalian target of rapamycin (mTOR) assembles a signaling network essential for the regulation of cell growth, which has emerged as a major target of anticancer therapies. The tuberous sclerosis complex 1 and 2 (TSC1/2) proteins and their target, the small GTPase Rheb, constitute a key regulatory pathway upstream of mTOR. Phospholipase D (PLD) and its product phosphatidic acid are also upstream regulators of the mitogenic mTOR signaling. However, how the TSC/Rheb and PLD pathways interact or integrate in the rapamycin-sensitive signaling network has not been examined before. Here, we find that PLD1, but not PLD2, is required for

Our observations reveal the involvement of PLD1 in mTOR signaling and cell size control, and provide a molecular mechanism for Cdc42 activation of S6K1. A new cascade is proposed to connect mitogenic signals to mTOR through Cdc42, PLD1, and PA.

SUMMARY Mice with targeted deletion of the growth hormone receptor (GHRKO mice) are GH resistant, small, obese, hypoinsulinemic, highly insulin sensitive and remarkably long-lived. To elucidate the unexpected coexistence of adiposity with improved insulin sensitivity and extended longevity, we examined effects of surgical removal of visceral (epididymal and perinephric) fat on metabolic traits related to insulin signaling and longevity. Comparison of results obtained in GHRKO mice and in normal animals from the same strain revealed disparate effects of visceral fat removal (VFR) on insulin and glucose tolerance, adiponectin levels, accumulation of ectopic fat, phosphorylation of insulin signaling intermediates, body temperature and respiratory quotient (RQ). Overall, VFR produced the expected improvements in insulin sensitivity and reduced body temperature and RQ in normal mice and had opposite effects in GHRKO mice. Some of the examined parameters were altered by VFR in opposite directions in GHRKO and normal mice, others were affected in only one genotype or exhibited significant genotype × treatment interactions. Functional differences between visceral fat of GHRKO and normal mice were confirmed by measurements of adipokine secretion, lipolysis and expression of genes related to fat metabolism. We conclude that in the absence of GH signaling the secretory activity of visceral fat is profoundly altered and unexpectedly promotes enhanced insulin sensitivity. The apparent beneficial effects of visceral fat in GHRKO mice may also explain why reducing adiposity by calorie restriction fails to improve insulin signaling or further extend longevity in these animals.

We examine the impact of targeted disruption of growth hormone-releasing hormone (GHRH) in mice on longevity and the putative mechanisms of delayed aging. GHRH knockout mice are remarkably long-lived, exhibiting major shifts in the expression of genes related to xenobiotic detoxification, stress resistance, and insulin signaling. These mutant mice also have increased adiponectin levels and alterations in glucose homeostasis consistent with the removal of the counter-insulin effects of growth hormone. While these effects overlap with those of caloric restriction, we show that the effects of caloric restriction (CR) and the GHRH mutation are additive, with lifespan of GHRH-KO mutants further increased by CR. We conclude that GHRH-KO mice feature perturbations in a network of signaling pathways related to stress resistance, metabolic control and inflammation, and therefore provide a new model that can be used to explore links between GHRH repression, downregulation of the somatotropic axis, and extended longevity.DOI: http://dx.doi.org/10.7554/eLife.01098.001

The immunosuppressant rapamycin, in complex with its cellular receptor FKBP12, targets the cellular protein FKBP12-rapamycin-associated protein/mammalian target of rapamycin/rapamycin and FKBP12 target 1 (FRAP/mTOR/RAFT1) and inhibits/delays G 1 cell cycle progression in mammalian cells. As a member of the novel phosphatidylinositol kinase-related kinase family, FRAP's kinase activity is essential for its signaling function. The FKBP12-rapamycin binding (FRB) domain in FRAP is also speculated to play an important role in FRAP function and signaling. However, the biochemical and physiological functions of FRB, as well as the mechanism for rapamycin inhibition, have been unclear. The present study focuses on investigation of FRB's role and the functional relationship between FRB domain and kinase domain in FRAP. Microinjection of purified FRB protein into human osteosarcoma MG63 cells results in a drastic blockage of the G 1 to S cell cycle progression; such a dominant negative effect is reversed by a point mutation (Trp 2027 3 Phe). The same mutation also abolishes kinase activity of FRAP without affecting ATP binding, and truncation studies suggest that upstream sequences including FRB are required for kinase activity in vitro. Given these data, we propose a model for FRAP function, in which the FRB domain is required for activation of the kinase domain, possibly through the interaction with an upstream activator. In addition, our observations provide direct evidence linking FRAP function to G 1 cell cycle progression.Mammalian cell proliferation is regulated by extracellular mitogens via multiple signal transduction pathways. One such pathway leads to the up-regulation of protein synthesis, which is essential for G 1 progression of the cell cycle (1-3). At least two proteins involved in regulating the translational machinery have been found to lie downstream of this pathway: the p70 S6 kinase (p70 s6k ) 1 (4, 5) and an eIF4E binding protein (4E-BP1) (3, 6 -8). The immunosuppressant rapamycin inhibits this pathway at a point upstream of p70 s6k (9, 10) and 4E-BP1 (11-14); this inhibition requires the presence of the cellular protein FKBP12 (15) and results in selective reduction of protein synthesis (16 -19) and G 1 arrest in a variety of mammalian cells (15), as well as in the yeast Saccharomyces cerevisiae (20,21).A major player in the rapamycin-sensitive pathway has been identified as the cellular target of rapamycin-FKBP12 complex, designated FRAP (22), RAFT1 (23), or mTOR (24). FRAP belongs to the novel family of phosphatidylinositol kinase (PIK)-related kinases which include Ataxia telangiectasia mutated. Members of this family are involved in a range of essential cellular functions, including cell cycle progression, cell cycle checkpoints, DNA repair, and DNA recombination (25-28). A kinase domain with sequence homology to lipid and protein kinases has been found at the C termini of all members in this family, and the kinase activity is crucial for the functions of these proteins. The FRAP protein is a 28...