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 acid- and 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. 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 acid- and 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) 2The abbreviations used are: mTORC1, mammalian target of rapamycin complex 1; 4EBP1, eukaryotic translation initiation factor 4E-binding protein 1; GAP, GTPase-activating protein; PKB, protein kinase B; RBD, Ras-binding domain; S6K, S6 kinase; siRNA, small interfering RNA; RNAi, RNA interference. functions as a critical intracellular component of the nutrient-sensing system and governs many aspects of cellular responses to changing nutrient levels (1.Avruch J. Hara K. Lin Y. Liu M. Long X. Ortiz-Vega S. Yonezawa K. Oncogene. 2006; 25: 6361-6372Crossref PubMed Scopus (269) Google Scholar, 2.Corradetti M.N. Guan K.L. Oncogene. 2006; 25: 6347-6360Crossref PubMed Scopus (337) Google Scholar, 3.Dann S.G. Thomas G. FEBS Lett. 2006; 580: 2821-2829Crossref PubMed Scopus (181) Google Scholar, 4.Guertin D.A. Sabatini D.M. Cancer Cell. 2007; 12: 9-22Abstract Full Text Full Text PDF PubMed Scopus (2482) Google Scholar, 5.Wullschleger S. Loewith R. Hall M.N. Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4826) Google Scholar). 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.Inoki K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2477) Google Scholar, 7.Potter C.J. Pedraza L.G. Xu T. Nat. Cell Biol. 2002; 4: 658-665Crossref PubMed Scopus (793) Google Scholar, 8.Castro A.F. Rebhun J.F. Clark G.J. Quilliam L.A. J. Biol. Chem. 2003; 278: 32493-32496Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 9.Zhang Y. Gao X. Saucedo L.J. Ru B. Edgar B.A. Pan D. Nat. Cell Biol. 2003; 5: 578-581Crossref PubMed Scopus (729) Google Scholar). It has been proposed that the decreased GAP activity allows Rheb to stay in an “active” GTP-bound 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.Roccio M. Bos J.L. Zwartkruis F.J. Oncogene. 2006; 25: 657-664Crossref PubMed Scopus (121) Google Scholar, 11.Long X. Lin Y. Ortiz-Vega S. Yonezawa K. Avruch J. Curr. Biol. 2005; 15: 702-713Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar, 12.Gulati P. Gaspers L.D. Dann S.G. Joaquin M. Nobukuni T. Natt F. Kozma S.C. Thomas A.P. Thomas G. Cell Metab. 2008; 7: 456-465Abs