Intracellular Sensing of Amino Acids in Xenopus laevis Oocytes Stimulates p70 S6 Kinase in a Target of Rapamycin-dependent Manner

Abstract

Amino acids exert modulatory effects on proteins involved in control of mRNA translation in animal cells through the target of rapamycin (TOR) signaling pathway. Here we use oocytes of Xenopus laevis to investigate mechanisms by which amino acids are "sensed" in animal cells. Small (ϳ48%) but physiologically relevant increases in intracellular but not extracellular total amino acid concentration (or Leu or Trp but not Ala, Glu, or Gln alone) resulted in increased phosphorylation of p70 S6K and its substrate ribosomal protein S6. This response was inhibited by rapamycin, demonstrating that the effects require the TOR pathway. Alcohols of active amino acids substituted for amino acids with lower efficiency. Oocytes were refractory to changes in external amino acid concentration unless surface permeability of the cell to amino acids was increased by overexpression of the System L amino acid transporter. Amino acid-induced, rapamycin-sensitive activation of p70 S6K was conferred when System L-expressing oocytes were incubated in extracellular amino acids, supporting intracellular localization of the putative amino acid sensor. In contrast to lower eukaryotes such as yeast, which possess an extracellular amino acid sensor, our findings provide the first direct evidence for an intracellular location for the putative amino acid sensor in animal cells that signals increased amino acid availability to TOR/p70 S6K .There is growing evidence that nutrients, in particular amino acids (AAs), 1 exert powerful modulatory effects on proteins involved in cell signaling, mRNA translation, and amino acid transport (for review, see Refs. 1-3). Recent studies indicate that signaling pathways responsive to AAs in mammalian cells show a similarity to those that have already been well characterized in lower organisms such as yeast and bacteria (4 -6). The serine-threonine protein kinase mammalian target of rapamycin ((m)TOR) is proposed to act as a "nutrient-dependent gatekeeper" (5) for several cell functions. Two key translational regulators, p70 S6 kinase (p70 S6K ) and eukaryotic initiation factor 4E-binding protein (4E-BP1) lie downstream of (m)TOR in a cell signaling pathway responsive to AA supplementation (7-10). Increased phosphorylation of p70 S6K and 4E-BP1 in response to AA supplementation is inhibited by the immunosuppressant rapamycin, a specific inhibitor of (m)TOR (5, 6). The activation of p70 S6K by phosphorylation enables it to phosphorylate its downstream target ribosomal protein S6 and increase translation of so-called 5Ј-terminal tract of pyrimidines mRNAs, which generally encode ribosomal proteins and elongation factors (2, 11). The activation of p70 S6K is thought to lead to up-regulation of ribosome biosynthesis and thus increase the translational capacity of the cell. Phosphorylation of 4E-BP1 increases protein synthesis by releasing eukaryotic initiation factor 4E, which can then form the eukaryotic initiation factor 4F complex as a prerequisite for translation of the capped mRNAs (reviewed in Ref. 2). Althoug...

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