Figure 6

Glutamine and serine selectively regulate PAT4-dependent rapamycin-resistant mTORC1 signalling in HCT116 cells. (a) HCT116 cells were starved of the essential amino acids, glycine (Gly), isoleucine (Ile), leucine (Leu), threonine (Thr) and tryptophan (Trp), and the non-essential amino acids, serine (Ser) and glutamine (Gln), for 4 h in separate cultures, and then proteins extracted and subjected to western blotting. After this time, glutamine depletion produces the greatest reduction in γ-phosphorylation of 4E-BP1 (arrow); serine depletion also induces a more modest shift. (b) Clones of HCT116 cells carrying the IPTG-inducible PAT4 shRNA constructs, shPAT4(4.8), and the IPTG-inducible non-targeting control construct, shNT, were exposed to culture medium containing different concentrations of glutamine for 4 h in the absence of IPTG. Under these conditions, shPAT4(4.8) cells express about 50% of normal PAT4 mRNA levels (Supplementary Figure S1B) because of leaky PAT4 shRNA transcription. As glutamine concentration falls, a greater reduction in γ-phosphorylation of 4E-BP1 is apparent in shPAT4(4.8) cells. S6 phosphorylation also seems to be affected at low glutamine concentrations. (c) Same experiment as (b), except different levels of serine in the culture medium. Again, shPAT4(4.8) cells have lower 4E-BP1 γ-phosphorylation at reduced concentrations of serine. (d and e) Rapamycin-treated HCT116 cells subjected to different levels of glutamine (d) and serine starvation (e), over 4 h, show a greater reduction in γ-phosphorylation of 4E-BP1.