mTOR pathway is activated by amino acids but does not affect amino acid-dependent proliferation.

<p>(A) Amino acid treatment increases mTor signaling in CD98/Lat-1 cells. Cells were serum deprived for 24 hours then subjected to amino acid deprivation for an additional two hours (time 0). Cells were then treated with serum free media containing a full complement of amino acids. Lysates were isolated at the time points indicated and subjected to SDS-PAGE. Immunoblots were probed with phos-p70S6K (Thr389) as well as antibodies recognizing the multiple phosphorylated forms of 4EBP1. (B) mTor pathways do not regulate amino acid-dependent proliferation of CD98/Lat-1 cells. Cells were plated onto 96-well plates, serum deprived for 24 hours then deprived of amino acids for an additional 2 hours and pretreated with Rapamycin (100 nM) and PP242 (2.5 µM) prior to addition of ³H-Thymidine in serum free media with a full complement of amino acids for an 8 hour pulse. ³H-Thymidine incorporation was determined as described in Experimental Procedures then. Values are the mean ± s.e.m. of a representative experiment performed in eight replicates. (C and D) Inhibitors of mTor, MAPK and PI3 kinase were effective in inhibiting amino acid-dependent signaling. (C) Cells were serum deprived for 24 hours then subjected to amino acid deprivation for an additional two hours (time 0) and pretreated with Rapamycin and PP242 . Cells were treated with serum free media containing a full complement of amino acids in the presence of inhibitors. Lysates were isolated after 2hours of amino acid treatment and subjected to SDS-PAGE. Immunoblots were probed with phos-p70S6K (Thr389) as well as an antibody recognizing the hyperphosphorylated forms of 4EBP1. (D) Cells were treated as described in C in the presence of LY294002, SB 203580 and U0126, all at 10 µM. Lysates were isolated after two hours of amino acid treatment in the presence of inhibitors and subjected to SDS-PAGE.