In response to amino acid availability, the class III PI-3-kinase hVps34 activates the phospholipase PLD and mTORC1 signaling to regulate mammalian cell size.

Phosphatidic acid (PA) is a critical mediator of mitogenic activation of mammalian target of rapamycin complex 1 (mTORC1) signaling, a master regulator of mammalian cell growth and proliferation. The mechanism by which PA activates mTORC1 signaling has remained unknown. Here, we report that PA selectively stimulates mTORC1 but not mTORC2 kinase activity in cells and in vitro. Furthermore, we show that PA competes with the mTORC1 inhibitor, FK506 binding protein 38 (FKBP38), for mTOR binding at a site encompassing the rapamycin-FKBP12 binding domain. This leads to PA antagonizing FKBP38 inhibition of mTORC1 kinase activity in vitro and rescuing mTORC1 signaling from FKBP38 in cells. Phospholipase D 1, a PA-generating enzyme that is an established upstream regulator of mTORC1, is found to negatively affect mTOR-FKBP38 interaction, confirming the role of endogenous PA in this regulation. Interestingly, removal of FKBP38 alone is insufficient to activate mTORC1 kinase and signaling, which require PA even when the FKBP38 level is drastically reduced by RNAi. In conclusion, we propose a dual mechanism for PA activation of mTORC1: PA displaces FKBP38 from mTOR and allosterically stimulates the catalytic activity of mTORC1.

SUMMARY The mammalian target of rapamycin complex 1 (mTORC1) is regulated, in part, by the endogenous inhibitor DEPTOR. However, the mechanism of DEPTOR regulation with regard to rapid mTORC1 activation remains unknown. We report that DEPTOR is rapidly and temporarily dissociated from mTORC1 upon mitogenic stimulation, suggesting a mechanism underlying acute mTORC1 activation. This mitogen-stimulated DEPTOR dissociation is blocked by inhibition or depletion of the mTORC1 regulator, phospholipase D (PLD), and recapitulated with the addition of the PLD product phosphatidic acid (PA). Our mass spectrometry analysis has independently identified DEPTOR as an mTOR binding partner dissociated by PA. Interestingly, only PA species with unsaturated fatty acid chains, such as those produced by PLD, are capable of displacing DEPTOR and activating mTORC1, with high affinity for the FRB domain of mTOR. Our findings reveal a novel mechanism of mTOR regulation and provide a molecular explanation for the exquisite specificity of PA function.

SUMMARY Amino acid availability activates signaling by the mammalian target of rapamycin (mTOR) complex 1, mTORC1, a master regulator of cell growth. The class III PI-3-kinase Vps34 mediates amino acid signaling to mTORC1 by regulating lysosomal translocation and activation of the phospholipase PLD1. Here we identify leucyl-tRNA synthetase (LRS) as a leucine sensor for the activation of Vps34-PLD1 upstream of mTORC1. LRS is necessary for amino acid-induced Vps34 activation, cellular PI(3)P level increase, PLD1 activation, and PLD1 lysosomal translocation. Leucine binding but not tRNA charging activity of LRS is required for this regulation. Moreover, LRS physically interacts with Vps34 in amino acid-stimulatable non-autophagic complexes. Finally, purified LRS protein activates Vps34 kinase in vitro in a leucine-dependent manner. Collectively, our findings provide compelling evidence for a direct role of LRS in amino acid activation of Vps34 via a non-canonical mechanism, and fill a gap in the amino acid-sensing mTORC1 signaling network.

Rictor, a component of mammalian target of rapamycin complex 2 (mTORC2), controls neutrophil chemotaxis by regulating the dynamics of the actin cytoskeleton via Rac and Cdc42. This function of Rictor is independent of mTORC2 and the kinase activity of mTOR.

investigation has revealed that PLD1 is unlikely to regulate myogenesis through modulation of the actin cytoskeleton as previously suggested. Instead, PLD1 positively regulates mTOR signaling leading to the production of IGF2, an autocrine factor instrumental for the initiation of satellite cell differentiation. Furthermore, exogenous IGF2 fully rescues the differentiation defect resulting from PLD1 knockdown. Hence, PLD1 is critically involved in skeletal myogenesis by regulating the mTOR-IGF2 pathway. Key words: mTOR, Myogenesis, Phospholipase D (PLD) Summary PLD regulates myoblast differentiation through the

Nutrient overload is associated with the development of obesity, insulin resistance, and type II diabetes. High plasma concentrations of amino acids have been found to correlate with insulin resistance. At the cellular level, excess amino acids impair insulin signaling, the mechanisms of which are not fully understood. Here, we report that STAT3 plays a key role in amino acid dampening of insulin signaling in hepatic cells. Excess amino acids inhibited insulin-stimulated Akt phosphorylation and glycogen synthesis in mouse primary hepatocytes as well as in human hepatocarcinoma HepG2 cells. STAT3 knockdown protected insulin sensitivity from inhibition by amino acids. Amino acids stimulated the phosphorylation of STAT3 at Ser 727 , but not Tyr 705 . Replacement of the endogenous STAT3 with wild-type, but not S727A, recombinant STAT3 restored the ability of amino acids to inhibit insulin signaling, suggesting that Ser 727 phosphorylation was critical for STAT3-mediated amino acid effect. Furthermore, overexpression of STAT3-S727D was sufficient to inhibit insulin signaling in the absence of excess amino acids. Our results also indicated that mammalian target of rapamycin was likely responsible for the phosphorylation of STAT3 at Ser 727 in response to excess amino acids. Finally, we found that STAT3 activity and the expression of its target gene socs3, known to be involved in insulin resistance, were both stimulated by excess amino acids and inhibited by rapamycin. In conclusion, our study reveals STAT3 as a novel mediator of nutrient signals and identifies a Ser 727 phosphorylation-dependent and Tyr 705 phosphorylation-independent STAT3 activation mechanism in the modulation of insulin signaling.Insulin resistance is a major risk factor and a principal defect in type II diabetes. Nutrient overload in affluent societies has been associated with increased occurrence of metabolic syndrome (1, 2). High protein diets are associated with altered glucose metabolism and increased occurrence of type II diabetes (3, 4). Elevated plasma concentrations of amino acids have long been found in obesity and insulin-resistant states (5-8). Furthermore, amino acid infusion induces insulin resistance in healthy individuals (9). Most recently, it has been reported that branched-chain amino acids in diet contribute to insulin resistance in high fat diet-fed rats and that a similar consequence of such a dietary pattern may exist in human (10). Currently, a role of dietary proteins in the pathogenesis of insulin resistance has been well recognized (11) (14); among them the mammalian target of rapamycin (mTOR) has been shown to phosphorylate STAT3 in neuronal cells (15, 16) and IL-6-stimulated hepatocytes (17).As a negative feedback control, STATs induce the expression of SOCS proteins, which are characterized by their ability to down-regulate cytokine signaling (18). SOCSs also play an important role in the pathogenesis of insulin resistance by integrating cytokine signaling with insulin signaling (19). Overexpression of SOCS3 inhibited ...

Mammalian target of rapamycin complex 2 (mTORC2) controls a wide range of cellular and developmental processes, but its regulation remains incompletely understood. Through a yeast twohybrid screen, we have identified XPLN (exchange factor found in platelets, leukemic, and neuronal tissues), a guanine nucleotide exchange factor (GEF) for Rho GTPases, as an interacting partner of mTOR. In mammalian cells, XPLN interacts with mTORC2 but not with mTORC1, and this interaction is dependent on rictor. Knockdown of XPLN enhances phosphorylation of the Ser/Thr kinase Akt, a target of mTORC2, whereas overexpression of XPLN suppresses it, suggesting that XPLN inhibits mTORC2 signaling to Akt. Consistent with Akt promoting cell survival and XPLN playing a negative role in this process, XPLN knockdown protects cells from starvation-induced apoptosis. Importantly, this effect of XPLN depletion is abolished by inhibition of Akt or mTOR kinase activity, as well as by rictor knockdown. In vitro, purified XPLN inhibits mTORC2 kinase activity toward Akt without affecting mTORC1 activity. Interestingly, the GEF activity of XPLN is dispensable for its regulation of mTORC2 and Akt in cells and in vitro, whereas an N-terminal 125-amino-acid fragment of XPLN is both necessary and sufficient for the inhibition of mTORC2. Finally, as a muscle-enriched protein, XPLN negatively regulates myoblast differentiation by suppressing mTORC2 and Akt, and this function is through the XPLN N terminus and independent of GEF activity. Our study identifies XPLN as an endogenous inhibitor of mTORC2 and delineates a noncanonical mechanism of XPLN action. M ammalian target of rapamycin (mTOR) is an evolutionarily conserved Ser/Thr kinase that integrates signals from nutrient availability, growth factors, differentiation inducers, and various types of stress to control a wide range of cellular and developmental processes (1, 2). mTOR nucleates two distinct multiprotein complexes known as mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), characterized by the presence of raptor and rictor, respectively. Emerging evidence implicates the deregulation of mTOR signaling in a variety of diseases including cancer and diabetes (1), underscoring the importance of fully understanding the regulation of mTOR signaling. mTORC1 regulates cell growth and proliferation by promoting biosynthesis of proteins, lipids, and organelles while inhibiting autophagy (1, 2). The best-characterized substrates for the mTORC1 kinase are S6 kinase 1 (S6K1) and eIF-4E-binding protein-1 (4E-BP1), both key regulators of protein synthesis (3). mTORC2 phosphorylates the hydrophobic motif site Ser473 on the Ser/Thr kinase Akt that is necessary for its activation (4), as well as the turn motif controlling the folding and stability of Akt (5, 6). The ribosome plays a direct role in activating mTORC2 (7), and association with the ribosome also allows mTORC2 to phosphorylate and stabilize Akt cotranslationally (8). Thus, mTORC2 is involved in a variety of processes that are regulated by Akt, in...