Figure 1.
UA inhibits leucine-stimulated mTOR activation via a PI3K–Akt-independent manner.
(A), Serum-starved C2C12 myotubes were pretreated with or without UA at the indicated concentrations for 60 min, followed with or without 10 nM insulin (Ins) for 10 min (A) or 10 mM leucine (Leu) for 60 min (C). The phosphorylation and protein levels of Akt, S6K, 4EBP1, and ERK1/2 were determined by Western blot with indicated antibodies. (B) and (D), S6K phosphorylation in (A) and (C) was semi-quantified using the NIH Image J Program and normalized to S6K protein levels. (E), Serum-starved wild type and PDK1-knockout (KO) MEFs were pretreated with or without 50 µM UA for 60 minutes, followed with or without 10 mM leucine (Leu) for 60 minutes. The phosphorylation and protein levels of the interesting signaling molecules were determined by Western blot using specific antibodies as indicated. (F), S6K phosphorylation in (E) was semi-quantified using the NIH Image J Program and normalized to S6K protein levels. Tubulin was used as a loading control for all experiments, differences between groups were examined for statistical significance using ANOVA. Data are presented as mean±S.E.M. from three independent experiments. *, P<0.05, **P<0.01, N, no addition.
Figure 2.
UA inhibits leucine-stimulated mTOR activation in a TSC1/2 and Rheb-independent manner.
(A), Serum-starved TSC1/2+/+ (WT) and TSC1/2−/− (KO) MEF cells were pretreated with or without 50 µM for 60 min and then treated with or without 10 mM leucine (Leu) for 60 min. The phosphorylation and protein levels of S6K, 4E-BP1 and the protein levels of TSC1 and TSC2 in cell lysates were determined by Western blot with the indicated antibodies. (B), S6K phosphorylation shown in (A) was semi-quantified and normalized to S6K protein levels. (C), serum-starved C2C12 cells transiently expressing Rheb or RhebS16H were treated with or without 50 µM UA for 1 hour, followed with or without 10 mM leucine (Leu) for 60 min. The phosphorylation and protein levels of S6K, and the protein levels of FLAG-Rheb in cell lysates were determined by Western blot using indicated antibodies. (D), S6K phosphorylation in (C) was semi-quantified using the NIH Image J Program and normalized with S6K protein levels. Differences between groups were examined for statistical significance using ANOVA. Data are presented as mean ± S.E.M. from three independent experiments. *, P<0.05, **, P<0.01, and ***, p<0.001. N, no addition.
Figure 3.
UA does not inhibit the associations between mTOR and Raptor or Deptor.
(A), Deptor-suppressed and scrambled C2C12 cells were serum starved and pretreated with or without 50 µM UA for 60 min, followed with or without 10 mM leucine (Leu) for 60 min. The phosphorylation of S6K, 4EBP1 and the protein levels of S6K, 4EBP1, Deptor, and Tubulin were determined by Western blot using specific antibodies. (B), S6K phosphorylation in (A) was semi-quantified using the NIH Image J Program and normalized with S6K protein levels. (C), C2C12 cells were serum starved and pretreated with or without leucine (Leu) for 60 min, followed with or without 50 µM UA for 60 min. mTOR was immunoprecipitated from cell lysates using an anti-mTOR antibody and the co-immunoprecipitaed Raptor or Deptor was determined by Western blot. Differences between groups were examined for statistical significance using ANOVA. Data are presented as mean ± S.E.M. from three independent experiments. NIg, normal immunoglobulin. *, P<0.05, **, P<0.01, and ***, p<0.001. N, no addition.
Figure 4.
UA inhibits leucine-stimulated mTORC1 activity by disrupting mTOR lysosomal localization.
(A), C2C12 cells transiently expressing control vector, RagB or RagBGTP were serum starved and treated with or without 50 µM UA for 60 min, followed with or without 10 mM leucine (Leu) for 60 min. The phosphorylation and protein levels of S6K and the protein levels of RagB, FLAG, and Tubulin in cell lysates were determined by Western blot using specific antibodies as indicated. (B), S6K phosphorylation in (A) was semi-quantified using the NIH Image J Program and normalized to the levels of S6K in cells. (C), C2C12 cells transiently expressing RagB were serum starved and pretreated with or without with or without 50 µM UA for 60 min, followed with or without 10 mM leucine (Leu) for 60 min. Cells were then treated with the cross-linker chemical DSP and lysed. Raptor was immunoprecipitated from cell lysates and its associated proteins were detected by Western blot. (D), The cellular levels of RagB in (C) was semi-quantified using the NIH Image J Program and normalized to Raptor. (E), Serum-starved C2C12 cells transiently expressing RagB were pretreated with or without leucine (Leu) for 60 min and then with 50 µM UA for 60 min. Cells were then treated with DSP and lysed. FLAG-taged RagB was immunoprecipitaed from cell lysates and the associated proteins were determined by Western blot using antibodies to mTOR or Raptor. (F), the levels of Raptor in (E) was semi-quantified using the NIH Image J Program and normalized to FLAG-RagB. Differences between groups were examined for statistical significance using ANOVA. Data are presented as mean ± S.E.M. from three independent experiments. NIg, normal immunoglobulin. *, P<0.05, **, P<0.01, and ***, P<0.001. N, no addition. (G), serum-starved C2C12 cells were pretreated with or without 50 µM UA for 60 min, followed with or without 10 mM leucine (Leu) for 60 min. Cells were stained using antibodies specific to mTOR (red) and LAMP1 (green) and visualized by a confocal immunofluorescence microscope. Inset: Higher magnification to show colocalization (yellow) of mTOR and LAMP1. Scale bar, 10 µm.