RalA Functions as an Indispensable Signal Mediator for the Nutrient-sensing System

Abstract

Cells sense nutrients present in the extracellular environment and modulate the activities of intracellular signaling systems in response to nutrient availability. This study demonstrates that RalA and its activator RalGDS participate in nutrient sensing and are indispensable for activation of mammalian target of rapamycin complex 1 (mTORC1) induced by extracellular nutrients. Knockdown of RalA or RalGDS abolished amino acidand glucose-induced mTORC1 activation, as judged by phosphorylation of S6 kinase and eukaryotic translation initiation factor 4E-binding protein 1. The amount of GTP-bound RalA increased in response to increased amino acid availability. In addition, RalA knockdown suppressed Rheb-induced S6 kinase phosphorylation, and the constitutively active form of RalA induced mTORC1 activation in the absence of Rheb. These results collectively suggest that RalGDS and RalA act downstream of Rheb and that RalA activation is a crucial step in nutrient-induced mTORC1 activation.Nutrients such as amino acids and carbohydrates are available in the extracellular space and are vital for cell homeostasis. Cells are able to detect extracellular nutrients and modulate their intracellular signaling systems in response to changes in nutrient levels. Recent studies have shown that mammalian target of rapamycin complex 1 (mTORC1) 2 functions as a critical intracellular component of the nutrient-sensing system and governs many aspects of cellular responses to changing nutrient levels (1-5). Signaling cascades initiated by growth factors and leading to mTORC1 activation have been extensively characterized. Growth factor stimulation results primarily in the phosphorylation of TSC2, a GTPase-activating protein (GAP) for Rheb GTPase, in a phosphoinositide 3-kinase-and protein kinase B (PKB)-dependent manner, followed by inactivation of TSC2 GAP activity (6 -9). It has been proposed that the decreased GAP activity allows Rheb to stay in an "active" GTPbound form, leading to mTORC1 activation. In contrast to the mechanism of the growth factor-initiated mTORC1 activation, little is known about the mechanism(s) by which mTORC1 activity is regulated by amino acid availability (10 -16). Recent studies have shown that calmodulin and hVps34 play a role in the amino acid-sensing system (12) and that Rag GTPases escort mTORC1 to sites where they can activate the kinase (17, 18). However, most of the amino acid-sensing signaling machinery, including the amino acid sensors themselves, is unknown, and the mechanism(s) underlying the nutrient-sensing process remains to be identified.RalA and RalB GTPases belong to the Ras superfamily and are known to participate in multiple cellular processes through binding to diverse effector proteins. RalA effectors described to date include Ral-binding protein 1, Sec5, Exo84, filamin, and ZO-1-associated nucleic acid-binding protein (19 -22); these interactions are indicative of crucial roles for RalA in cell migration, membrane dynamics, and transcriptional regulation. RalB is highly homologous to R...

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

“…We and others (31) observed that recombinant FKBP38 inhibited mTORC1 signaling in cells only when it was highly overexpressed, which may explain the absence of the effect of FKBP38 in similar experiments performed by some groups (33,34). The requirement of high levels of recombinant FKBP38 to FIGURE 6.…”

“…Because FKBP38 binds mTOR through a region that overlaps with the PA-binding FRB domain (13,14), it appeared plausible that PA could compete with FKBP38 for mTOR binding as a mechanism of activating mTORC1. However, although several groups independently demonstrated a role of FKBP38 as a negative regulator of mTORC1 (13,31,32), others challenged this conclusion (33,34). Therefore, we deemed it necessary to reexamine the role of FKBP38 in mTORC1 signaling in the Chen laboratory.…”

FKBPs and the Akt/mTOR pathway

“…23 The larger FKBP38 has been proposed to be an endogenous inhibitor of mTOR by binding to mTOR independently of rapamycin in a manner displaceable by the mTOR activator Rheb. [24][25][26][27] However, the biochemical interaction of FKBP38 with Rheb or mTOR as well as its role in the cellular regulation of mTOR could not be replicated by several other groups, [28][29][30][31] and the role of FKBP38 in mTOR regulation remains controversial.…”

Mammalian Target of Rapamycin (mTOR): Conducting the Cellular Signaling Symphony

“…Several GTPases (e.g. RAGs and RalA) have also been shown to promote mTORC1 signaling in response to amino acid sufficiency (61)(62)(63). A recent model posits that GTP-loaded RAG heterodimers bind raptor and induce the recruitment of mTORC1 from an ill defined cytoplasmic compartment to a Rab7-positive late endosomal membrane compartment that contains the mTORC1 activator Rheb (62).…”