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. 2008 Dec 12;283(50):35053-9.
doi: 10.1074/jbc.M805822200. Epub 2008 Oct 23.

RalA functions as an indispensable signal mediator for the nutrient-sensing system

Tomohiko Maehama  1 , Masahiko Tanaka, Hiroshi Nishina, Makoto Murakami, Yasunori Kanaho, Kentaro Hanada
Affiliations

RalA functions as an indispensable signal mediator for the nutrient-sensing system

Tomohiko Maehama et al. J Biol Chem. .

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.

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Figures

FIGURE 1.
FIGURE 1.
Ral GTPase knockdown inhibits nutrient-induced mTORC1 activation. HeLa cells were transfected with siRNAs against the indicated targets. After 2 days in culture, cells were deprived of amino acids (A), glucose (B), or serum (C) for 2 h. Cells were then exposed to amino acids (A), glucose (B), or 2 μg/ml bovine insulin (C) for the indicated times, followed by immunoblot analysis. D, quantification of the phospho-S6K (normalized to total S6K) immunoblots shown in A-C, as described under “Materials and Methods.” Values represent the mean ± S.D. of 3-5 independent experiments. Calculated p values (Student's t test) are presented.
FIGURE 2.
FIGURE 2.
RalGDS plays an indispensable role in nutrient-induced mTORC1 activation. A and B, HeLa cells were transfected with siRNAs, as indicated. After 2 days in culture, cells were deprived of amino acids for 2 h and then exposed to amino acids for the indicated times, followed by immunoblot analysis (B). The original unprocessed gel image is presented in supplemental Fig. 6. The parallel experiment to confirm RalGDS knockdown was also performed by reverse transcription-PCR analysis (A). C, HeLa cells were transfected with siRNAs, as indicated. After 2 days in culture, cells were deprived of amino acids, glucose, or serum for 2 h. Cells were then exposed to amino acids, glucose, or 2 μg/ml bovine insulin for the indicated times, followed by immunoblot analysis. D, quantification of the phospho-S6K (normalized to total S6K) immunoblots shown in C, as described under “Materials and Methods.” Values represent the mean ± S.D. of 3-5 independent experiments.
FIGURE 3.
FIGURE 3.
RalA is activated in response to amino acid availability, and the activation is required for nutrient-induced mTORC1 activation. A and B, HeLa cells were deprived of amino acids for 2 h, followed by exposure to amino acids for the indicated times. The RalA activation assay was then performed as described under “Materials and Methods.” Values represent the mean ± S.D. of four independent experiments. Calculated p values (Student's t test) are presented. C, HeLa cells were transfected with the DNA construct as indicated. After 1 day in culture, cells were deprived of amino acids or serum for 2 h. Cells were then exposed to amino acids or 2 μg/ml bovine insulin for the indicated times followed by immunoblot analysis. D, quantification of the phospho-S6K (normalized to total S6K) immunoblots shown in C, as described under “Materials and Methods.” Values represent the mean ± S.D. of 3-5 independent experiments.
FIGURE 4.
FIGURE 4.
RalA functions downstream of Rheb. A, HeLa cells were transfected with control siRNA or siRNA against RalA. After 1 day in culture, cells were further transfected with empty pCAGGS or Rheb[S16H]/pCAGGGS plasmid DNA. After 1 day in culture, cells were deprived of amino acids for 2 h. Cells were then exposed to amino acids for the indicated times, followed by immunoblot analysis. B, quantification of the phospho-S6K (normalized to total S6K) immunoblot shown in A, as described under “Materials and Methods.” Bars represent differences between two independent experiments. C, HeLa cells were transfected with control siRNA or siRNA against Rheb. After 1 day in culture, cells were further transfected with empty pCMV5 or RalA[Q72L]/pCMV5 plasmid DNA. After 1 day in culture, cells were deprived of amino acids for 2 h. Cells were then exposed to amino acids for the indicated times, followed by immunoblot analysis. Quantification of the phospho-S6K (normalized to total S6K) immunoblot was performed as described under “Materials and Methods.” The first row of lane description in the figure represents averages of and differences between duplicate determinations in a typical study. D, HeLa cells were transfected with equal amounts of RalA expression construct (pCMV5 empty vector or RalA[Q72L]/pCMV5) and Rheb expression construct (pCAGGS empty vector or Rheb[S20N]/pCAGGS). After 1 day in culture, cells were deprived of amino acids for 2 h. Cells were then exposed to amino acids for the indicated times followed by immunoblot analysis. Quantification of the phospho-S6K (normalized to total S6K) immunoblot was performed as described under “Materials and Methods.” The first row of lane description in the figure represents averages of and differences between duplicate determinations in a typical study. Original unprocessed gel images are presented in supplemental Fig. 6.
FIGURE 5.
FIGURE 5.
Model for the action of RalGDS and RalA. Extracellular nutrients activate RalGDS to promote guanine nucleotide exchange of RalA. Activated RalA then facilitates the action of Rheb to activate mTORC1 in a FKBP38-independent manner. In contrast, growth factor stimulation activates Rheb primarily through the inactivation of TSC2. Rheb then activates mTORC1 in a RalA-independent manner.
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