Phospholipase D Mediates Nutrient Input to Mammalian Target of Rapamycin Complex 1 (mTORC1)

Abstract

The mammalian target of rapamycin (mTOR) is a critical sensor of nutritional sufficiency. Although much is known about the regulation of mTOR in response to growth factors, much less is known about the regulation of mTOR in response to nutrients. Amino acids have no impact on the signals that regulate Rheb, a GTPase required for the activation of mTOR complex 1 (mTORC1). Phospholipase D (PLD) generates a metabolite, phosphatidic acid, that facilitates association between mTOR and the mTORC1 co-factor Raptor. We report here that elevated PLD activity in human cancer cells is dependent on both amino acids and glucose and that amino acid-and glucose-induced increases in mTORC1 activity are dependent on PLD. Amino acid-and glucose-induced PLD and mTORC1 activity were also dependent on the GTPases RalA and ARF6 and the type III phosphatidylinositol-3-kinase hVps34. Thus, a key stimulatory event for mTORC1 activation in response to nutrients is the generation of phosphatidic acid by PLD.The mammalian target of rapamycin (mTOR) 2 is a critical regulator of cell growth and cell cycle progression. mTOR is activated in response to both growth factors and nutrients (1). mTOR exists as two complexes, mTORC1 and mTORC2. Although both mTORC1 and mTORC2 are responsive to growth factors, mTORC1 is believed to be the primary sensor of nutrient and energy sufficiency (2-4). mTORC1 is activated in response to insulin and other peptide hormones that activate type I phosphatidylinositol (PI) 3-kinases (PI3Ks) that generate PI 3,4,5-trisphosphate. The activation of mTORC1 via PI3K involves the Akt-mediated suppression of the tuberous sclerosis complex (TSC), which consists of TSC1 and TSC2. TSC1/2 functions as a GTPase-activating protein for Rheb (Ras homologue enriched in brain), a GTPase that directly interacts with and contributes to the activation of mTORC1 (2). Although Rheb is required for the amino acid stimulation of mTORC1, starving cells of amino acids has no effect on GTP loading (4 -7). In addition, amino acid starvation was still able to suppress mTORC1 in cells lacking the Rheb GTPase-activating complex protein TSC2 (4 -7). Therefore, although there is a requirement for GTP-bound Rheb for induction of mTORC1 by amino acids, amino acids do not impact on Rheb activity, indicating that regulation of Rheb does not stimulate mTORC1 in response to amino acids.It was recently reported that Rag GTPases are critical for targeting mTORC1 to lysosomal compartments in response to amino acids (8 -10), and a model was proposed whereby the targeting of mTOR to lysosomal membranes containing GTPbound Rheb was sufficient to activate mTORC1 (10, 11). However, several other factors implicated in the regulation of mTORC1 in response to amino acids were not accounted for in this model. The Ras family GTPase RalA was reported to be required for amino acid induction of mTORC1 (12). Significantly, RalA is constitutively associated with phospholipase D1 (PLD1) (13,14). PLD1 generates the lipid second messenger phosphatidic acid (PA), which is r...

Phospholipase D and the Maintenance of Phosphatidic Acid Levels for Regulation of Mammalian Target of Rapamycin (mTOR)

“…Much has been learned in the last several years on the mechanistic basis for the sensing of amino acids by mTOR at the lysosomal membrane via Rag GTPases (27,41). The activation of mTOR in response to amino acids also requires PLD (19,20,42). However, very little is known about the dependence of mTOR on glucose, another critical nutrient sensed by mTOR.…”

“…It is also of note that forced localization of mTOR to lysosomes activated mTOR in the absence of amino acids if Rheb was present (69). Rheb is one of many GTPases that activate PLD1 (20,70,71), indicating that PLD may work in concert with the signaling mechanisms that activate Rheb. The picture that emerges is one where LPAAT-generated PA may be the more critical source for nutrient sensing by mTOR, but that PLD is the more versatile source of PA that can respond locally to growth factor/insulin signals and stress.…”

“…However, growth factors (6) and nutrients (19,20) also stimulate PA production through the action of phospholipases that breakdown membrane phospholipids, potentially leading to high PA concentrations at specific locations and times. This can be accomplished by PLD, or a combination of phospholipase C (PLC), which generates DG, and the subsequent conversion to PA by DGK.…”

The RAS-RAL axis in cancer: evidence for mutation-specific selectivity in non-small cell lung cancer

“…PLD1 is another important effector of RAL GTPase; PLD1 plays a role in membrane trafficking and as a signaling molecule [50] . PLD1 is responsible for RALA-mediated mTOR pathway activation [51] . RALA acts as a nutrient sensor and promotes protumorigenic signaling through mTOR with the help of PLD1.…”

Protein Phosphorylation as a Key Mechanism of mTORC1/2 Signaling Pathways

“…Therefore, besides PA ability to activate mTORC1, there are several data indicating requirement of PLD itself for the activation of mTORC1. Very recent study provided additional evidence that nutrient stimulation of mTORC1 is dependent on PLD activity which in turn is activated by small GTPases RalA and Arf6 [127]. According to this study, amino acids dependent activation of PLD is mediated trough generated by Vps34 PI-3-phosphate [127], that could interact with PX domains of PLD1 and PLD2 which are known to be critical for PLD activity [128].…”