Mee Sup Yoon

Phospholipase D1 is an effector of Rheb in the mTOR pathway.

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 Rheb activation of the mTOR pathway, as demonstrated by the effects of RNAi. The overexpression of Rheb activates PLD1 in cells in the absence of mitogenic stimulation, and the knockdown of Rheb impairs serum stimulation of PLD activation. Furthermore, the overexpression of TSC2 suppresses PLD1 activation, whereas the knockdown or deletion of TSC2 leads to elevated basal activity of PLD. Consistent with a TSC-Rheb-PLD signaling cascade, AMPK and PI3K, both established regulators of TSC2, appear to lie upstream of PLD as revealed by the effects of pharmacological inhibitors, and serum activation of PLD is also dependent on amino acid sufficiency. Finally, Rheb binds and activates PLD1 in vitro in a GTP-dependent manner, strongly suggesting that PLD1 is a bona fide effector for Rheb. Hence, our findings reveal an unexpected interaction between two cascades in the mTOR signaling pathways and open up additional possibilities for targeting this important growth-regulating network for the development of anticancer drugs.

Phosphatidic Acid Activates Mammalian Target of Rapamycin Complex 1 (mTORC1) Kinase by Displacing FK506 Binding Protein 38 (FKBP38) and Exerting an Allosteric Effect

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.

Signal Transducer and Activator of Transcription 3 (STAT3) Mediates Amino Acid Inhibition of Insulin Signaling through Serine 727 Phosphorylation

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 Ser727, but not Tyr705. 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 Ser727 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 Ser727 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 Ser727 phosphorylation-dependent and Tyr705 phosphorylation-independent STAT3 activation mechanism in the modulation of insulin signaling.

PLD regulates myoblast differentiation through the mTOR-IGF2 pathway

A mammalian target of rapamycin (mTOR) pathway is essential for the differentiation of cultured skeletal myoblasts in response to growth factor withdrawal. Previously, phospholipase D (PLD) has been found to play a role in cell growth regulation and mitogenic activation of mTOR signaling. However, a role for PLD in the autocrine regulation of myoblast differentiation is not known. Here we show that upon induction of differentiation in mouse C2C12 satellite cells, the expression of both PLD1 and PLD2 is upregulated. C2C12 differentiation is markedly inhibited by 1-butanol, an inhibitor of the PLD-catalyzed transphosphatidylation reaction, and also by the knockdown of PLD1, but not PLD2. Further 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.

Rapid Mitogenic Regulation of the mTORC1 Inhibitor, DEPTOR, by Phosphatidic Acid

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 mechanism of mTOR regulation and provide a molecular explanation for the exquisite specificity of PA function.

Mammalian Target of Rapamycin and Rictor control neutrophil chemotaxis by regulating Rac/Cdc42 activity and the actin cytoskeleton

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.

Distinct amino acid-sensing mTOR pathways regulate skeletal myogenesis

Amino acid–sensing mTOR signaling controls the homeostasis of skeletal myogenesis. The Rag GTPases negatively regulate differentiation by activating mTORC1 and subsequently suppressing the IRS1-PI3K-Akt pathway, whereas a Vps34-PLD1-PA-mTOR pathway activates the transcriptional regulation of Igf2 that is essential for myogenesis.

Mechanistic target of rapamycin controls homeostasis of adipogenesis

Signaling mediated by the mechanistic target of rapamycin (mTOR) is believed to play a critical and positive role in adipogenesis, based on pharmacological evidence and genetic manipulation of mTOR regulators and targets. However, there is no direct genetic evidence for an autonomous role of mTOR itself in preadipocyte differentiation. To seek such evidence, we employed a conditional knockdown approach to deplete mTOR in preadipocytes. Surprisingly, while knockdown of S6K1, a target of mTOR, impairs 3T3-L1 preadipocyte differentiation, reduction of mTOR levels leads to increased differentiation. This enhanced adipogenesis requires the remaining mTOR activity, as mTOR inhibitors abolish differentiation in the mTOR knockdown cells. We also found that mTOR knockdown elevates the levels of CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ). Furthermore, partial reduction of mTOR levels alleviates inhibition of Akt by mTORC1 via IRS1, while at the same time maintaining its positive input through mTORC1 into the adipogenic program. The greater sensitivity of the IRS1-Akt pathway to mTOR levels provides a mechanism that explains the net outcome of enhanced adipogenesis through PPARγ upon mTOR knockdown. Our observations reveal an unexpected role of mTOR in suppressing adipogenesis and suggest that mTOR governs the homeostasis of the adipogenic process by modulating multiple signaling pathways.

Raptor and Rheb Negatively Regulate Skeletal Myogenesis through Suppression of Insulin Receptor Substrate 1 (IRS1)

The mammalian target of rapamycin (mTOR) is essential for skeletal myogenesis through controlling distinct cellular pathways. The importance of the canonical mTOR complex 1 signaling components, including raptor, S6K1, and Rheb, had been suggested in muscle maintenance, growth, and metabolism. However, the role of those components in myogenic differentiation is not entirely clear. In this study we have investigated the functions of raptor, S6K1, and Rheb in the differentiation of C2C12 mouse myoblasts. We find that although mTOR knockdown severely impairs myogenic differentiation as expected, the knockdown of raptor, as well as Rheb, enhances differentiation. Consistent with a negative role for these proteins in myogenesis, overexpression of raptor or Rheb inhibits C2C12 differentiation. On the other hand, neither knockdown nor overexpression of S6K1 has any effect. Moreover, the enhanced differentiation elicited by raptor or Rheb knockdown is accompanied by increased Akt activation, elevated IRS1 protein levels, and decreased Ser-307 (human Ser-312) phosphorylation on IRS1. Finally, IRS1 knockdown eliminated the enhancement in differentiation elicited by raptor or Rheb knockdown, suggesting that IRS1 is a critical mediator of the myogenic functions of raptor and Rheb. In conclusion, the Rheb-mTOR/raptor pathway negatively regulates myogenic differentiation by suppressing IRS1-PI3K-Akt signaling. These findings underscore the versatility of mTOR signaling in biological regulations and implicate the existence of novel mTOR complexes and/or signaling mechanism in skeletal myogenesis.

XPLN is an endogenous inhibitor of mTORC2

Significance Mammalian target of rapamycin complex 2 (mTORC2) controls a wide range of cellular and developmental processes and is a potential target for therapeutic strategies against a range of human diseases. Here we identify XPLN (exchange factor found in platelets, leukemic, and neuronal tissues) as an endogenous inhibitor of mTORC2 and show that XPLN negatively regulates cell survival and skeletal myoblast differentiation by inhibiting mTORC2 and subsequently the Ser/Thr kinase Akt. This XPLN action requires its N terminus and is independent of its canonical activity as a guanine nucleotide exchange factor. Our findings provide a molecular understanding of mTORC2 regulation and uncover XPLN as a potentially important player in many aspects of biology and diseases involving mTORC2 and Akt. 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 two-hybrid 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.