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.
Highlights
Nutrients such as amino acids and carbohydrates are available in the extracellular space and are vital for cell homeostasis
We show that RalA is activated in response to extracellular amino acids and that both RalGDS and RalA are indispensable for nutrient-induced mammalian target of rapamycin complex 1 (mTORC1) activation
RalA Is an Indispensable Component of the Nutrient-sensing System—Cell proliferation is a dynamic event that requires highly coordinated multiple cellular processes, such as DNA replication and protein synthesis, which are controlled by growth factor-initiated growth/proliferation signals and nutrient-sensing signals. mTORC1 is a critical component in the organization of these signaling systems to regulate cell proliferation [1,2,3,4,5]
Summary
Cell Culture and Transfection—HeLa cells (ATCC CCL-2) were maintained as described previously [29]. 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] to generate Rheb/ pCAGGS. 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 5Ј-end, followed by cloning into the EcoRI/SalI site of pCMV5 The coding region of Sec5-RBD was amplified from the Sec5-RBD/pGEX vector [32] (gift of Dr Hisanori Horiuchi; Kyoto University) and tagged with a FLAG sequence at the 5Ј-end by PCR, followed by cloning into the BamHI/EcoRI site of pEF1-MycHisA (Invitrogen) to generate Sec5-RBD/pEF1-FLAG. Band intensity of the RalA blot was measured using Image J, as described under “Immunoblot.” Typical images from repeated experiments are represented. Glutathione S-transferase-fused Sec5-RBD protein was prepared as described previously [32], with the exception that Escherichia coli strain BL21(DE3) Codon Plus RP (Clontech) was used
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