Fig. 3.

Insensitivity of the mTORC1 pathway to amino acid deprivation in cells stably expressing RagB^GTP. (A) Cell size distributions (graphs) and S6K1 phosphorylation (immunoblot) of cells stably expressing RagB, Rheb1, Rag^GTP, or Rap2A. Mean cell diameters ± S.D. (μm) are: Rap2A, 16.05 ± 0.07; Rheb1, 16.79 ± 0.06; RagB, 16.40 ± 0.08; and RagB^GTP, 16.68 ± 0.06 (n = 4 and p < 0.0008 for all comparisons to Rap2A-expressing cells). HEK-293T cells transduced with lentiviruses encoding the specified proteins were deprived for 50 minutes for serum and (B) leucine or (C) total amino acids, and, where indicated, re-stimulated with leucine or amino acids for 10 minutes. Cell lysates were analyzed for the levels of the specified proteins and the phosphorylation state of S6K1. (D) Amino acid-stimulated interaction of the Rag proteins with mTORC1. HEK-293T cells stably expressing FLAG-tagged RagB, RagD, or RagB^GTP were starved for amino acids and serum for 50 minutes and, where indicated, re-stimulated with amino acids for 10 minutes. Cells were then processed with a chemical cross-linking assay and cell lysates and FLAG-immunoprecipitates analyzed for the levels of the indicated proteins. (E) Effects of amino acid stimulation on GTP loading of RagB. Values are mean ± s.d. for n = 3 (p < 0.02 for increase in GTP loading caused by amino acid stimulation). (F) Abundance of RagA, RagB, RagC, and RagD in HeLa cells expressing the indicated shRNAs. (G) S6K1 phosphorylation in HeLa cells expressing shRNAs targeting RagC and RagD. Cells were deprived of serum and leucine for 50 minutes, and, where indicated, re-stimulated with leucine for 10 minutes. (H) Effects of dsRNA-mediated knockdowns of Drosophila orthologues of RagB or RagC on amino acid-induced phosphorylation of dS6K.