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

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. 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Gaspers L.D. Dann S.G. Joaquin M. Nobukuni T. Natt F. Kozma S.C. Thomas A.P. Thomas G. Cell Metab. 2008; 7: 456-465Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar) and that Rag GTPases escort mTORC1 to sites where they can activate the kinase (17.Kim E. Goraksha-Hicks P. Li L. Neufeld T.P. Guan K.L. Nat. Cell Biol. 2008; 10: 935-945Crossref PubMed Scopus (1038) Google Scholar, 18.Sancak Y. Peterson T.R. Shaul Y.D. Lindquist R.A. Thoreen C.C. Bar-Peled L. Sabatini D.M. Science. 2008; 320: 1496-1501Crossref PubMed Scopus (2050) Google Scholar). 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.Cantor S.B. Urano T. Feig L.A. Mol. Cell. Biol. 1995; 15: 4578-4584Crossref PubMed Scopus (262) Google Scholar, 20.Moskalenko S. Henry D.O. Rosse C. Mirey G. Camonis J.H. White M.A. Nat. Cell Biol. 2002; 4: 66-72Crossref PubMed Scopus (356) Google Scholar, 21.Ohta Y. Suzuki N. Nakamura S. Hartwig J.H. Stossel T.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2122-2128Crossref PubMed Scopus (380) Google Scholar, 22.Frankel P. Aronheim A. Kavanagh E. Balda M.S. Matter K. Bunney T.D. Marshall C.J. EMBO J. 2005; 24: 54-62Crossref PubMed Scopus (92) Google Scholar); these interactions are indicative of crucial roles for RalA in cell migration, membrane dynamics, and transcriptional regulation. RalB is highly homologous to RalA and shares an identical effector domain with RalA. However, these two proteins are believed to have distinct functions in cells (23.Lim K.H. Baines A.T. Fiordalisi J.J. Shipitsin M. Feig L.A. Cox A.D. Der C.J. Counter C.M. Cancer Cell. 2005; 7: 533-545Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 24.Lim K.H. O'Hayer K. Adam S.J. Kendall S.D. Campbell P.M. Der C.J. Counter C.M. Curr. Biol. 2006; 16: 2385-2394Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 25.Shipitsin M. Feig L.A. Mol. Cell. Biol. 2004; 24: 5746-5756Crossref PubMed Scopus (113) Google Scholar); RalB participates in apoptotic signaling, whereas RalA is predominantly involved in proliferation-related signaling, particularly anchorage-independent proliferation (26.Chien Y. White M.A. EMBO Rep. 2003; 4: 800-806Crossref PubMed Scopus (161) Google Scholar, 27.Bodemann B.O. White M.A. Nat. Rev. Cancer. 2008; 8: 133-140Crossref PubMed Scopus (195) Google Scholar). In addition, an accumulating body of evidence shows that both RalA and RalGDS, an activator of RalA, are crucial for Ras-induced oncogenic transformation of cells and that Ras in fact binds to RalGDS and induces RalA activation (23.Lim K.H. Baines A.T. Fiordalisi J.J. Shipitsin M. Feig L.A. Cox A.D. Der C.J. Counter C.M. Cancer Cell. 2005; 7: 533-545Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 24.Lim K.H. O'Hayer K. Adam S.J. Kendall S.D. Campbell P.M. Der C.J. Counter C.M. Curr. Biol. 2006; 16: 2385-2394Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 26.Chien Y. White M.A. EMBO Rep. 2003; 4: 800-806Crossref PubMed Scopus (161) Google Scholar, 28.Gonzalez-Garcia A. Pritchard C.A. Paterson H.F. Mavria G. Stamp G. Marshall C.J. Cancer Cell. 2005; 7: 219-226Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). These observations suggest that the RalGDS-RalA signaling unit plays a role in cell proliferation; however, how RalA controls cell proliferation and participates in oncogenic transformation remains unknown. In this report, we show that RalA is activated in response to extracellular amino acids and that both RalGDS and RalA are indispensable for nutrient-induced mTORC1 activation. The RalGDS-RalA signaling unit functions downstream of Rheb and participates selectively in the nutrient-sensing system. Cell Culture and Transfection—HeLa cells (ATCC CCL-2) were maintained as described previously (29.Okahara F. Itoh K. Nakagawara A. Murakami M. Kanaho Y. Maehama T. Mol. Biol. Cell. 2006; 17: 4888-4895Crossref PubMed Scopus (73) Google Scholar). Amino acid-free RPMI1640 medium (30.Ijichi C. Matsumura T. Tsuji T. Eto Y. Biochem. Biophys. Res. Commun. 2003; 303: 59-64Crossref PubMed Scopus (97) Google Scholar) and glucose-free RPMI1640 medium (Invitrogen) were used for amino acid and glucose deprivation, respectively. Amino acid and glucose stimulation were performed by the addition of normal RPMI1640 medium and RPMI1640 medium containing 25 mm glucose, respectively. Serum starvation was carried out in RPMI1640 medium containing 0.1% fatty acid-free bovine serum albumin (Sigma). Transfection of plasmid DNA was carried out using either FuGENE HD (Roche Applied Science) or Lipofectamine 2000 (Invitrogen). Lipofectamine RNAi Max (Invitrogen) was used for transfection of siRNA; a mixed siRNA mixture was used unless otherwise specified. Transfections were performed according to the manufacturers' protocols. Plasmids and siRNAs—SMARTpool siRNA against human Rheb was purchased from Dharmacon. Other siRNAs (Stealth RNAi), including negative control siRNA, were purchased from Invitrogen: RALA RNAi 1 (HSS109033), RALA RNAi 3 (HSS109035), RALB RNAi 1-3 (HSS109036 to -109038), RALGDS RNAi 1 (HSS109039), and RALGDS RNAi 2 (HSS109040). Full-length human Rheb (NP_005605, amino acids 1-184) cDNA was amplified from HeLa cell cDNA by PCR and cloned into the EcoRI site of pCAGGS (31.Niwa H. Yamamura K. Miyazaki J. Gene (Amst.). 1991; 108: 193-199Crossref PubMed Scopus (4659) Google Scholar) to generate Rheb/pCAGGS. Rheb mutations S16H (tct to cat) and S20N (tcc to aat) were introduced by site-directed mutagenesis using a PCR-based strategy. Full-length human RalA (NP_005393, amino acids 1-206) cDNA was amplified from HeLa cell cDNA by PCR with the addition of Myc tag sequence at the 5′-end, followed by cloning into the EcoRI/SalI site of pCMV5 (gift from David W. Russell) to generate Myc-RalA/pCMV5. The Q72L mutation (cag to ctc) was introduced by site-directed mutagenesis employing a PCR-based strategy. The coding region of Sec5-RBD was amplified from the Sec5-RBD/pGEX vector (32.Kawato M. Shirakawa R. Kondo H. Higashi T. Ikeda T. K. S. T. H. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar) (gift of and with a sequence at the by followed by cloning into the site of (Invitrogen) to generate DNA was used for PCR and were using the were as described previously (29.Okahara F. Itoh K. Nakagawara A. Murakami M. Kanaho Y. Maehama T. Mol. Biol. Cell. 2006; 17: 4888-4895Crossref PubMed Scopus (73) Google Scholar). used were of the which to of the Signaling and The of and were using the the of a or control was the control was from as the of to was The were to that of the control and the were in from are in was from cells using (Invitrogen) and into cDNA using In to the RNAi against RalGDS, PCR was in a mixture using of cDNA as and RalGDS were and were and RalA was as described previously (32.Kawato M. Shirakawa R. Kondo H. Higashi T. Ikeda T. K. S. T. H. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar) with HeLa cells were in of 25 mm mm 1 mm mm 1 mm and mixture of the was with for were by and that were to Sec5-RBD protein were to the and for to GTP-bound RalA to to the were with and proteins and were to of the RalA was using as described from are were with bovine serum albumin to to were carried out at unless otherwise specified. Sec5-RBD protein was as described previously (32.Kawato M. Shirakawa R. Kondo H. Higashi T. Ikeda T. K. S. T. H. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google with the that was RalA an of the proliferation is a that highly multiple cellular such as DNA and protein which are by growth factor-initiated and nutrient-sensing mTORC1 is a critical component in the of these signaling systems to cell proliferation (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). In to the of GTPases in the signaling we GTPases using siRNA and the growth and nutrient-induced mTORC1 activation in HeLa cells of both RalA and RalB as by using and It is of that the used in this study both RalA and however, the shows for RalB for RalA to the phosphorylation of human at as a of mTORC1 activity this the has been as a mTORC1 phosphorylation site B.A. N. Kozma S.C. Thomas G. Mol. Cell. Biol. 17: PubMed Scopus Google Scholar, A. J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, M. N. P. D. Thomas G. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). cells were in amino phosphorylation of was to an 2 phosphorylation the of amino acids and a in HeLa cells RalA knockdown phosphorylation induced by the addition of amino whereas knockdown of RalB a of of phosphorylation 1, and was two siRNAs were used to RalA that the of mTORC1 activation was to an of the This was by the in which an RalA mutations at and phosphorylation of RalA knockdown Knockdown of either RalA or RalB glucose-induced phosphorylation that were of to for amino 1, B and that both the amino acid- and systems the signaling In cells were by which is known to activate mTORC1 in a phosphoinositide manner, RalA knockdown in of phosphorylation, and RalB knockdown in 1, and It is that of the cells in the of for in a of phosphorylation that was with that in a with either amino acids or This by the RalA knockdown be a of the for a nutrient for growth mTORC1 activation as described previously (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). In addition, the of RalA and RalB knockdown the phosphorylation of 4EBP1, for mTORC1 activation, were to their phosphorylation 1, These results suggest that the nutrient-sensing system GTPases to activate mTORC1 and that RalA is an indispensable whereas the phosphoinositide mTORC1 activation to a The the RalGDS-RalA Signaling is a activator of the GTPases in of its for cell used RNAi to RalGDS in HeLa cells in to its role in the amino acid-sensing system. we were to the knockdown using available to detect however, two distinct siRNAs against RalGDS of the RalGDS Cells with these siRNAs phosphorylation at the addition of amino acids to the medium GTPases to participate in the nutrient-induced mTORC1 activation we the RalGDS was for the nutrient-sensing system. shown in and results of the RalGDS knockdown were with for RalA knockdown of amino acid- and glucose-induced mTORC1 These suggest that RalGDS participates in the nutrient-sensing as an activator of In mTORC1 activation was by RalGDS knockdown and that induces mTORC1 activation in a Y. R. Feig L.A. Mol. Cell. Biol. 2008; PubMed Scopus Google Scholar) that RalGDS a complex with PKB, a critical component in growth mTORC1 activation, and activation in cell are able to the and the results of this study or the in the for RalA and RalGDS in mTORC1 activation. However, these results suggest that RalGDS has diverse signaling systems RalA and a mechanism for RalA RalA in to Amino that knockdown of either RalA or RalGDS, which of RalA, the amino acid-sensing system to RalA was activated in response to amino acid availability. RalA activation in cells using the Sec5-RBD which selectively binds to the GTP-bound form of GTPases can be used as an “active” (32.Kawato M. Shirakawa R. Kondo H. Higashi T. Ikeda T. K. S. T. H. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). and show that levels of the GTP-bound form of RalA increased a of cells with amino a In RalB activation was these In addition, of which by binding active RalA, suppressed amino phosphorylation of and In phosphorylation was by Sec5-RBD and the of these we that nutrient-induced activation of RalA is a critical step for the activation of RalA as a of is the small that plays a crucial role in the mTORC1 activation the of Rheb remains an accumulating body of evidence Rheb of and in to mTORC1 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, X. D. Liu A. X. Liu Y. Y. Science. 2007; PubMed Scopus Google Scholar, X. Ortiz-Vega S. Lin Y. Avruch J. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). is RalA or downstream of Rheb in the nutrient-sensing RalA its diverse such as and membrane (19.Cantor S.B. Urano T. Feig L.A. Mol. Cell. Biol. 1995; 15: 4578-4584Crossref PubMed Scopus (262) Google Scholar, 20.Moskalenko S. Henry D.O. Rosse C. Mirey G. Camonis J.H. White M.A. Nat. Cell Biol. 2002; 4: 66-72Crossref PubMed Scopus (356) Google Scholar, 21.Ohta Y. Suzuki N. Nakamura S. Hartwig J.H. Stossel T.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2122-2128Crossref PubMed Scopus (380) Google Scholar, 22.Frankel P. Aronheim A. Kavanagh E. Balda M.S. Matter K. Bunney T.D. Marshall C.J. EMBO J. 2005; 24: 54-62Crossref PubMed Scopus (92) Google which the nutrient In a the is involved in S. Henry D.O. Rosse C. Mirey G. Camonis J.H. White M.A. Nat. Cell Biol. 2002; 4: 66-72Crossref PubMed Scopus (356) Google Scholar, A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (113) Google Scholar, K. S. K. A. K. Y. Nat. Cell Biol. 2002; 4: PubMed Scopus Google Scholar); an plays a role in of the glucose to the membrane M. L. J. A.R. 2003; PubMed Scopus Google Scholar, M. L. A.R. Mol. Biol. Cell. 2006; 17: PubMed Scopus (97) Google Scholar). we the of RalA and RalGDS knockdown nutrient and that knockdown of amino acids or glucose of the Rheb S16H in a in phosphorylation and with a L. R. J. Y. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar). RalA knockdown in of phosphorylation in cells and the that RalA functions downstream of Rheb. In to this we RalA induced mTORC1 activation in the absence of Rheb. Knockdown of Rheb abolished amino mTORC1 activation, and of the constitutively active mTORC1 activation The negative amino mTORC1 activation by and the activity In addition, be that RalA RalGDS knockdown the of Rheb These observations collectively suggest that RalA primarily functions as a downstream component of Rheb in the amino acid-sensing system and that RalA Rheb to activation of RalA and Rheb in mTORC1 how RalA the from Rheb to mechanism is activation of mTORC1 by however, we were to detect activation of mTORC1 by RalA protein we were able to detect of either RalA or RalGDS with mTORC1 These results that RalA be to mTORC1 activation, and be This is by the that phosphorylation in cells Rheb was these results suggest that RalA the of Rheb to activate mTORC1 in response to extracellular study be to the underlying Rheb was shown to to and activate mTORC1 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, X. Ortiz-Vega S. Lin Y. Avruch J. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar) In addition, a X. D. Liu A. X. Liu Y. Y. Science. 2007; PubMed Scopus Google Scholar) that Rheb binds to an of mTORC1, and from mTORC1 we RalA to mTORC1 this we the and however, we were to detect the of these in HeLa cells In addition, be that we the of mTORC1 and phosphorylation by of knockdown of phosphorylation These observations are by a X. H. Liu R. A. Proud C.G. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google which that mTORC1 these results we that RalA participates in mTORC1 to Growth factor signaling cascades include Rheb as an indispensable of mTORC1 However, in contrast to the by nutrient-sensing growth mTORC1 activation TSC2, a as a critical A. Zwartkruis F.J. Nobukuni T. Joaquin M. Roccio M. H. Kozma S.C. E. Bos J.L. Thomas G. Mol. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, K. Li Y. Xu T. Guan K.L. 2003; 17: PubMed Scopus Google Scholar). the mechanism by which cells and growth Rheb remains unknown, be to a mechanism for the of RalA in mTORC1 activation into a However, that growth mTORC1 activation RalA that which is activated as a of growth factor stimulation and a distinct from the to activate activate both and to its L. R. J. Y. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google an for the that of RalA in of phosphorylation in cells and of RalA as a of the and RalA are known to be oncogenic factors that are for Ras-induced transformation of of studies have that these two are for the transformation (23.Lim K.H. Baines A.T. Fiordalisi J.J. Shipitsin M. Feig L.A. Cox A.D. Der C.J. Counter C.M. Cancer Cell. 2005; 7: 533-545Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 24.Lim K.H. O'Hayer K. Adam S.J. Kendall S.D. Campbell P.M. Der C.J. Counter C.M. Curr. Biol. 2006; 16: 2385-2394Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 26.Chien Y. White M.A. EMBO Rep. 2003; 4: 800-806Crossref PubMed Scopus (161) Google Scholar, 28.Gonzalez-Garcia A. Pritchard C.A. Paterson H.F. Mavria G. Stamp G. Marshall C.J. Cancer Cell. 2005; 7: 219-226Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar); however, the for the RalGDS-RalA has been to This study has that the RalGDS-RalA signaling unit plays a critical role in nutrient-sensing systems that mTORC1 activation in the of diverse intracellular In to a of cells of extracellular such as amino acids for protein and carbohydrates as a In addition, of intracellular such as protein and be however, of these systems these to the for RalA and RalGDS in cell proliferation and oncogenic studies be to the of RalGDS and RalA in the of RalGDS-RalA in the mTORC1 activation into roles of RalGDS and RalA in cell proliferation and oncogenic are to of for amino acid-free medium and to and for the Sec5-RBD/pGEX vector and for the RalA activation with