Insulin-like peptides and the target of rapamycin pathway coordinately regulate blood digestion and egg maturation in the mosquito Aedes aegypti

Background

Mosquitoes are insects that vector many serious pathogens to humans and other vertebrates. Most mosquitoes must feed on the blood of a vertebrate host to produce eggs. In turn, multiple cycles of blood feeding promote frequent contacts with hosts and make mosquitoes ideal disease vectors. Both hormonal and nutritional factors are involved in regulating egg development in the mosquito, Aedes aegypti. However, the processes that regulate digestion of the blood meal remain unclear.

Methodology/Principal Findings

Here we report that insulin peptide 3 (ILP3) directly stimulated late phase trypsin-like gene expression in blood fed females. In vivo knockdown of the mosquito insulin receptor (MIR) by RNA interference (RNAi) delayed but did not fully inhibit trypsin-like gene expression in the midgut, ecdysteroid (ECD) production by ovaries, and vitellogenin (Vg) expression by the fat body. In contrast, in vivo treatment with double-stranded MIR RNA and rapamycin completely blocked egg production. In vitro experiments showed that amino acids did not simulate late phase trypsin-like gene expression in the midgut or ECD production by the ovaries. However, amino acids did enhance ILP3-mediated stimulation of trypsin-like gene expression and ECD production.

Conclusions/Significance

Overall, our results indicate that ILPs from the brain synchronize blood meal digestion and amino acid availability with ovarian ECD production to maximize Vg expression by the fat body. The activation of digestion by ILPs may also underlie the growth promoting effects of insulin and TOR signaling in other species.     

Target of rapamycin-dependent activation of S6 kinase is a central step in the transduction of nutritional signals during egg development in a mosquito

Female mosquitoes are effective disease vectors, because they take blood from vertebrate hosts to obtain nutrients for egg development. Amino acid signaling via the target of rapamycin (TOR) pathway has been identified as a key requirement for the activation of egg development after a blood meal. We report the characterization of the TOR kinase and one of its major downstream targets, S6 kinase, of the yellow fever mosquito Aedes aegypti during egg development in adult females. Both TOR and S6K mRNA are expressed at high levels in the ovaries and in lower levels in fat body and other tissues. After a blood meal, the subcellular localization of TOR shifts from the cytoplasm to the plasma membrane of fat body cells. By detecting phosphothreonine 388 of mosquito S6 kinase, we show that TOR activity strongly increases in fat body and ovaries after a blood meal in vivo. Furthermore, phosphorylation of S6 kinase increases in in vitro cultured fat bodies after stimulation with amino acids. This increase is sensitive to the TOR inhibitor rapamycin in a concentration-dependent manner but not to the phosphatidylinositol 3-kinase/phosphatidylinositol 3-kinase-related kinase inhibitor LY294002, the MAPK inhibitor PD98059, or the translational inhibitor cycloheximide. RNA interference-mediated reduction of S6 kinase strongly inhibits the amino acid-induced up-regulation of the major yolk protein vitellogenin in vitro and effectively disrupts egg development after a blood meal in vivo. Our data show that TOR-dependent activation of S6 kinase is a central step in the transduction of nutritional information during egg development in mosquitoes.     

A Critical Role of the Nuclear Receptor HR3 in Regulation of Gonadotrophic Cycles of the Mosquito Aedes aegypti

The orphan nuclear receptor HR3 is essential for developmental switches during insect development and metamorphosis regulated by 20-hydroxyecdysone (20E). Reproduction of female mosquitoes of the major vector of Dengue fever, Aedes aegypti, is cyclic because of its dependence on blood feeding. 20E is an important hormone regulating vitellogenic events in this mosquito; however, any role for HR3 in 20E-driven reproductive events has not been known. Using RNA interference (RNAi) approach, we demonstrated that Aedes HR3 plays a critical role in a timely termination of expression of the vitellogenin (Vg) gene encoding the major yolk protein precursor. It is also important for downregulation of the Target-of-Rapamycin pathway and activation of programmed autophagy in the Aedes fat body at the end of vitellogenesis. HR3 is critical in activating betaFTZ-F1, EcRB and USPA, the expressions of which are highly elevated at the end of vitellogenesis. RNAi depletion of HR3 (iHR3) prior to the first gonadotrophic cycle affects a normal progression of the second gonadotrophic cycle. Most of ovaries 24 h post second blood meal from iHR3 females in the second cycle were small with follicles that were only slightly different in length from of those of resting stage. In addition, these iHR3 females laid a significantly reduced number of eggs per mosquito as compared to those of iMal and the wild type. Our results indicate an important role of HR3 in regulation of 20E-regulated developmental switches during reproductive cycles of A. aegypti females.     

Programmed autophagy in the fat body of Aedes aegypti is required to maintain egg maturation cycles

Autophagy plays a pivotal role by allowing cells to recycle cellular components under conditions of stress, starvation, development and cancer. In this work, we have demonstrated that programmed autophagy in the mosquito fat body plays a critical role in maintaining of developmental switches required for normal progression of gonadotrophic cycles. Mosquitoes must feed on vertebrate blood for their egg development, with each gonadotrophic cycle being tightly coupled to a separate blood meal. As a consequence, some mosquito species are vectors of pathogens that cause devastating diseases in humans and domestic animals, most importantly malaria and Dengue fever. Hence, deciphering mechanisms to control egg developmental cycles is of paramount importance for devising novel approaches for mosquito control. Central to egg development is vitellogenesis, the production of yolk protein precursors in the fat body, the tissue analogous to a vertebrate liver, and their subsequent specific accumulation in developing oocytes. During each egg developmental cycle, the fat body undergoes a developmental program that includes previtellogenic build-up of biosynthetic machinery, intense production of yolk protein precursors, and termination of vitellogenesis. The importance of autophagy for termination of vitellogenesis was confirmed by RNA interference (RNAi) depletions of several autophagic genes (ATGs), which inhibited autophagy and resulted in untimely hyper activation of TOR and prolonged production of the major yolk protein precursor, vitellogenin (Vg). RNAi depletion of the ecdysone receptor (EcR) demonstrated its activating role of autophagy. Depletion of the autophagic genes and of EcR led to inhibition of the competence factor, betaFTZ-F1, which is required for ecdysone-mediated developmental transitions. Moreover, autophagy-incompetent female mosquitoes were unable to complete the second reproductive cycle and exhibited retardation and abnormalities in egg maturation. Thus, our study has revealed a novel function of programmed autophagy in maintaining egg maturation cycles in mosquitoes.     

SLC7 amino acid transporters of the yellow fever mosquito Aedes aegypti and their role in fat body TOR signaling and reproduction

BackgroundAn important function of the fat body in adult female mosquitoes is the conversion of blood meal derived amino acids (AA) into massive amounts of yolk protein precursors. A highly efficient transport mechanism for AAs across the plasma membrane of the fat body trophocytes is essential in order to deliver building blocks for the rapid synthesis of large amounts of these proteins. This mechanism consists in part of AA transporter proteins from the solute carrier family. These transporters have dual function; they function as transporters and participate in the nutrient signal transduction pathway that is activated in the fat body after a blood meal. In this study we focused on the solute carrier 7 family (SLC7), a family of AA transporters present in all metazoans that includes members with strong substrate specificity for cationic AAs.Methodology/principal findingsWe identified 11 putative SLC7 transporters in the genome sequence of Aedes aegypti. Phylogenetic analysis puts five of these in the cationic AA transporter subfamily (CAT) and six in the heterodimeric AA transporter (HAT) subfamily. All 11 A. aegypti SLC7 genes are expressed in adult females. Expression profiles are dynamic after a blood meal. We knocked down six fat body-expressed SLC7 transporters using RNAi and found that these ‘knockdowns’ reduced AA-induced TOR signaling. We also determined the effect these knockdowns had on the number of eggs deposited following a blood meal.Conclusions/significanceOur analysis stresses the importance of SLC7 transporters in TOR signaling pathway and mosquito reproduction.     

Rheb (Ras Homologue Enriched in Brain)-dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Activation Becomes Indispensable for Cardiac Hypertrophic Growth after Early Postnatal Period

Cardiomyocytes proliferate during fetal life but lose their ability to proliferate soon after birth and further increases in cardiac mass are achieved through an increase in cell size or hypertrophy. Mammalian target of rapamycin complex 1 (mTORC1) is critical for cell growth and proliferation. Rheb (Ras homologue enriched in brain) is one of the most important upstream regulators of mTORC1. Here, we attempted to clarify the role of Rheb in the heart using cardiac-specific Rheb-deficient mice (Rheb^−/−). Rheb^−/− mice died from postnatal day 8 to 10. The heart-to-body weight ratio, an index of cardiomyocyte hypertrophy, in Rheb^−/− was lower than that in the control (Rheb^+/+) at postnatal day 8. The cell surface area of cardiomyocytes isolated from the mouse hearts increased from postnatal days 5 to 8 in Rheb^+/+ mice but not in Rheb^−/− mice. Ultrastructural analysis indicated that sarcomere maturation was impaired in Rheb^−/− hearts during the neonatal period. Rheb^−/− hearts exhibited no difference in the phosphorylation level of S6 or 4E-BP1, downstream of mTORC1 at postnatal day 3 but showed attenuation at postnatal day 5 or 8 compared with the control. Polysome analysis revealed that the mRNA translation activity decreased in Rheb^−/− hearts at postnatal day 8. Furthermore, ablation of eukaryotic initiation factor 4E-binding protein 1 in Rheb^−/− mice improved mRNA translation, cardiac hypertrophic growth, sarcomere maturation, and survival. Thus, Rheb-dependent mTORC1 activation becomes essential for cardiomyocyte hypertrophic growth after early postnatal period.     

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.     

Targeted gene expression in the transgenic Aedes aegypti using the binary Gal4-UAS system

In this study, we report the establishment of the binary Gal4/UAS system for the yellow fever mosquito Aedes aegypti. We utilized the 1.8-kb 5′ upstream region of the vitellogenin gene (Vg) to genetically engineer mosquito lines with the Vg-Gal4 activator and established UAS-EGFP responder transgenic mosquito lines to evaluate the binary Gal4/UAS system. The results show that the Vg-Gal4 driver leads to a high level of tissue-, stage- and sex-specific expression of the EGFP reporter in the fat body of Vg-Gal4/UAS-EGFP hybrids after blood-meal activation. In addition, the applicability of this system to study hormonal regulation of gene expression was demonstrated in in vitro organ culture experiments in which the EGFP reporter was highly activated in isolated fat bodies of previtellogenic Vg-Gal4/UAS-EGFP females incubated in the presence of 20-hydroxyecdysone (20E). Hence, this study has opened the door for further refinement of genetic tools in mosquitoes.     

Expression of budding yeast FKBP12 confers rapamycin susceptibility to the unicellular red alga Cyanidioschyzon merolae

The target of rapamycin (TOR) is serine/threonine protein kinase that is highly conserved among eukaryotes and can be inactivated by the antibiotic rapamycin through the formation of a ternary complex composed of rapamycin and two proteins, TOR and FKBP12. Differing from fungi and animals, plant FKBP12 proteins are unable to form the ternary complex, and thus plant TORs are insensitive to rapamycin. This has led to a poor understanding of TOR functions in plants. As a first step toward the understanding of TOR function in a rapamycin-insensitive unicellular red alga, Cyanidioschyzon merolae, we constructed a rapamycin-susceptible strain in which the Saccharomyces cerevisiae FKBP12 protein (ScFKBP12) was expressed. Treatment with rapamycin resulted in growth inhibition and decreased polysome formation in this strain. Binding of ScFKBP12 with C. merolae TOR in the presence of rapamycin was demonstrated in vivo and in vitro by pull-down experiments. Moreover, in vitro kinase assay showed that inhibition of C. merolae TOR kinase activity was dependent on ScFKBP12 and rapamycin.     

Dynamic Switch of Negative Feedback Regulation in Drosophila Akt–TOR Signaling

Akt represents a nodal point between the Insulin receptor and TOR signaling, and its activation by phosphorylation controls cell proliferation, cell size, and metabolism. The activity of Akt must be carefully balanced, as increased Akt signaling is frequently associated with cancer and as insufficient Akt signaling is linked to metabolic disease and diabetes mellitus. Using a genome-wide RNAi screen in Drosophila cells in culture, and in vivo analyses in the third instar wing imaginal disc, we studied the regulatory circuitries that define dAkt activation. We provide evidence that negative feedback regulation of dAkt occurs during normal Drosophila development in vivo. Whereas in cell culture dAkt is regulated by S6 Kinase (S6K)–dependent negative feedback, this feedback inhibition only plays a minor role in vivo. In contrast, dAkt activation under wild-type conditions is defined by feedback inhibition that depends on TOR Complex 1 (TORC1), but is S6K–independent. This feedback inhibition is switched from TORC1 to S6K only in the context of enhanced TORC1 activity, as triggered by mutations in tsc2. These results illustrate how the Akt–TOR pathway dynamically adapts the routing of negative feedback in response to the activity load of its signaling circuit in vivo.     

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.     

Rheb binds and regulates the mTOR kinase

BACKGROUND: The target of rapamycin (TOR), in complex with the proteins raptor and LST8 (TOR complex 1), phosphorylates the p70S6K and 4E-BP1 to promote mRNA translation. Genetic evidence establishes that TOR complex activity in vivo requires the small GTPase Rheb, and overexpression of Rheb can rescue TOR from inactivation in vivo by amino-acid withdrawal. The Tuberous Sclerosis heterodimer (TSC1/TSC2) functions as a Rheb GTPase activator and inhibits TOR signaling in vivo. RESULTS: Here, we show that Rheb binds to the TOR complex specifically, independently of its ability to bind TSC2, through separate interactions with the mTOR catalytic domain and with LST8. Rheb binding to the TOR complex in vivo and in vitro does not require Rheb guanyl nucleotide charging but is modulated by GTP and impaired by certain mutations (Ile39Lys) in the switch 1 loop. Nucleotide-deficient Rheb mutants, although capable of binding mTOR in vivo and in vitro, are inhibitory in vivo, and the mTOR polypeptides that associate with nucleotide-deficient Rheb in vivo lack kinase activity in vitro. Reciprocally, mTOR polypeptides bound to Rheb(Gln64Leu), a mutant that is nearly 90% GTP charged, exhibit substantially higher protein kinase specific activity than mTOR bound to wild-type Rheb. CONCLUSIONS: The TOR complex 1 is a direct target of Rheb-GTP, whose binding enables activation of the TOR kinase.     

Acceleration of autoimmune diabetes in Rheb-congenic NOD mice with β-cell-specific mTORC1 activation

The protein Ras homolog enriched in brain (Rheb) is a Ras-like small GTPase that activates the mechanistic target of rapamycin complex 1 (mTORC1), which promotes cell growth. We previously generated transgenic C57BL/6 mice overexpressing Rheb in β-cells (B6^Rheb), which exhibited increased β-cell size and improved glucose tolerance with higher insulin secretion than wild type C57BL/6 mice. The mice also showed resistance to obesity-induced hyperglycemia, a model of type 2 diabetes, and to multiple low-dose-streptozotocin (MLDS)-induced hyperglycemia, a model of type 1 diabetes (T1D). To investigate whether the effects of mTORC1 activation by Rheb in B6^Rheb mice would also be evident in NOD mice, a spontaneous autoimmune T1D model, we created two NOD mouse lines overexpressing Rheb in their β-cells (NOD^Rheb; R3 and R20). We verified Rheb overexpression in β-cells, the relative activation of mTORC1 and β-cell enlargement. By 35 weeks of age, diabetes incidence was significantly greater in the R3 line and tended to be greater in the R20 line than in NOD mice. Histological analysis demonstrated that insulitis was significantly accelerated in 12-week-old R3 NOD^Rheb mice compared with NOD mice. Furthermore, serum insulin autoantibody (IAA) expression was significantly higher than that of NOD mice. We also examined whether complete Freund’s adjuvant (CFA) treatment alone or with glucagon-like peptide-1 (GLP-1) analog would reverse the hyperglycemia of NOD^Rheb mice; unexpectedly, almost none achieved normoglycemia. In summary, diabetes progression was significantly accelerated rather than prevented in NOD^Rheb mice. Our results suggest that the β-cell enlargement might merely enhance the autoimmunity of pathogenic T-cells against islets, leading to acceleration of autoimmune diabetes. We conclude that not only enlargement but also regeneration of β-cells in addition to the prevention of β-cell destruction will be required for the ideal therapy of autoimmune T1D.     

The Major Yolk Protein Vitellogenin Interferes with the Anti-Plasmodium Response in the Malaria Mosquito Anopheles gambiae

When taking a blood meal on a person infected with malaria, female Anopheles gambiae mosquitoes, the major vector of human malaria, acquire nutrients that will activate egg development (oogenesis) in their ovaries. Simultaneously, they infect themselves with the malaria parasite. On traversing the mosquito midgut epithelium, invading Plasmodium ookinetes are met with a potent innate immune response predominantly controlled by mosquito blood cells. Whether the concomitant processes of mosquito reproduction and immunity affect each other remains controversial. Here, we show that proteins that deliver nutrients to maturing mosquito oocytes interfere with the antiparasitic response. Lipophorin (Lp) and vitellogenin (Vg), two nutrient transport proteins, reduce the parasite-killing efficiency of the antiparasitic factor TEP1. In the absence of either nutrient transport protein, TEP1 binding to the ookinete surface becomes more efficient. We also show that Lp is required for the normal expression of Vg, and for later Plasmodium development at the oocyst stage. Furthermore, our results uncover an inhibitory role of the Cactus/REL1/REL2 signaling cassette in the expression of Vg, but not of Lp. We reveal molecular links that connect reproduction and immunity at several levels and provide a molecular basis for a long-suspected trade-off between these two processes.     

Specific Activation of mTORC1 by Rheb G-protein in Vitro Involves Enhanced Recruitment of Its Substrate Protein

Rheb G-protein plays critical roles in the TSC/Rheb/mTOR signaling pathway by activating mTORC1. The activation of mTORC1 by Rheb can be faithfully reproduced in vitro by using mTORC1 immunoprecipitated by the use of anti-raptor antibody from mammalian cells starved for nutrients. The low in vitro kinase activity against 4E-BP1 of this mTORC1 preparation is dramatically increased by the addition of recombinant Rheb. On the other hand, the addition of Rheb does not activate mTORC2 immunoprecipitated from mammalian cells by the use of anti-rictor antibody. The activation of mTORC1 is specific to Rheb, because other G-proteins such as KRas, RalA/B, and Cdc42 did not activate mTORC1. Both Rheb1 and Rheb2 activate mTORC1. In addition, the activation is dependent on the presence of bound GTP. We also find that the effector domain of Rheb is required for the mTORC1 activation. FKBP38, a recently proposed mediator of Rheb action, appears not to be involved in the Rheb-dependent activation of mTORC1 in vitro, because the preparation of mTORC1 that is devoid of FKBP38 is still activated by Rheb. The addition of Rheb results in a significant increase of binding of the substrate protein 4E-BP1 to mTORC1. PRAS40, a TOR signaling (TOS) motif-containing protein that competes with the binding of 4EBP1 to mTORC1, inhibits Rheb-induced activation of mTORC1. A preparation of mTORC1 that is devoid of raptor is not activated by Rheb. Rheb does not induce autophosphorylation of mTOR. These results suggest that Rheb induces alteration in the binding of 4E-BP1 with mTORC1 to regulate mTORC1 activation.Rheb defines a unique member of the Ras superfamily G-proteins (1). We have shown that Rheb proteins are conserved and are found from yeast to human (2). Although yeast and fruit fly have one Rheb, mouse and human have two Rheb proteins termed Rheb1 (or simply Rheb) and Rheb2 (RhebL1) (2). Structurally, these proteins contain G1-G5 boxes, short stretches of amino acids that define the function of the Ras superfamily G-proteins including guanine nucleotide binding (1, 3, 4). Rheb proteins have a conserved arginine at residue 15 that corresponds to residue 12 of Ras (1). The effector domain required for the binding with downstream effectors encompasses the G2 box and its adjacent sequences (1, 5). Structural analysis by x-ray crystallography further shows that the effector domain is exposed to solvent, is located close to the phosphates of GTP especially at residues 35–38, and undergoes conformational change during GTP/GDP exchange (6). In addition, all Rheb proteins end with the CAAX (C is cysteine, A is an aliphatic amino acid, and X is the C-terminal amino acid) motif that signals farnesylation. In fact, we as well as others have shown that these proteins are farnesylated (7–9).Rheb plays critical roles in the TSC/Rheb/mTOR signaling, a signaling pathway that plays central roles in regulating protein synthesis and growth in response to nutrient, energy, and growth conditions (10–14). Rheb is down-regulated by a TSC1·TSC2 complex that acts as a GTPase-activating protein for Rheb (15–19). Recent studies established that the GAP domain of TSC2 defines the functional domain for the down-regulation of Rheb (20). Mutations in the Tsc1 or Tsc2 gene lead to tuberous sclerosis whose symptoms include the appearance of benign tumors called hamartomas at different parts of the body as well as neurological symptoms (21, 22). Overexpression of Rheb results in constitutive activation of mTOR even in the absence of nutrients (15, 16). Two mTOR complexes, mTORC1 and mTORC2, have been identified (23, 24). Whereas mTORC1 is involved in protein synthesis activation mediated by S6K and 4EBP1, mTORC2 is involved in the phosphorylation of Akt in response to insulin. It has been suggested that Rheb is involved in the activation of mTORC1 but not mTORC2 (25).Although Rheb is clearly involved in the activation of mTOR, the mechanism of activation has not been established. We as well as others have suggested a model that involves the interaction of Rheb with the TOR complex (26–28). Rheb activation of mTOR kinase activity using immunoprecipitated mTORC1 was reported (29). Rheb has been shown to interact with mTOR (27, 30), and this may involve direct interaction of Rheb with the kinase domain of mTOR (27). However, this Rheb/mTOR interaction is a weak interaction and is not dependent on the presence of GTP bound to Rheb (27, 28). Recently, a different model proposing that FKBP38 (FK506-binding protein 38) mediates the activation of mTORC1 by Rheb was proposed (31, 32). In this model, FKBP38 binds mTOR and negatively regulates mTOR activity, and this negative regulation is blocked by the binding of Rheb to FKBP38. However, recent reports dispute this idea (33).To further characterize Rheb activation of mTOR, we have utilized an in vitro system that reproduces activation of mTORC1 by the addition of recombinant Rheb. We used mTORC1 immunoprecipitated from nutrient-starved cells using anti-raptor antibody and have shown that its kinase activity against 4E-BP1 is dramatically increased by the addition of recombinant Rheb. Importantly, the activation of mTORC1 is specific to Rheb and is dependent on the presence of bound GTP as well as an intact effector domain. FKBP38 is not detected in our preparation and further investigation suggests that FKBP38 is not an essential component for the activation of mTORC1 by Rheb. Our study revealed that Rheb enhances the binding of a substrate 4E-BP1 with mTORC1 rather than increasing the kinase activity of mTOR.     

The role of MAP4K3 in lifespan regulation of Caenorhabditiselegans

The TOR pathway is a kinase signaling pathway that regulates cellular growth and proliferation in response to nutrients and growth factors. TOR signaling is also important in lifespan regulation - when this pathway is inhibited, either naturally, by genetic mutation, or by pharmacological means, lifespan is extended. MAP4K3 is a Ser/Thr kinase that has recently been found to be involved in TOR activation. Unexpectedly, the effect of this protein is not mediated via Rheb, the more widely known TOR activation pathway. Given the role of TOR in growth and lifespan control, we looked at how inhibiting MAP4K3 in Caenorhabditiselegans affects lifespan. We used both feeding RNAi and genetic mutants to look at the effect of MAP4K3 deficiency. Our results show a small but significant increase in mean lifespan in MAP4K3 deficient worms. MAP4K3 thus represents a new target in the TOR pathway that can be targeted for pharmacological intervention to control lifespan.