Figure 2

PRL-3-driven mTOR activation correlates with increased Akt-TSC2-Rheb signalling.

(a) HCT116 cells overexpressing EGFP vector only (Vec), EGFP-tagged wild-type PRL-3 (PRL-3) or EGFP-tagged catalytic-inactive PRL-3 C104S (C104S) were cultured for 24 h in full media under normoxia (Ctrl), hypoxia (Hx), or serum-free (SF) conditions, prior to lysis and western blot analysis with the indicated antibodies. (b) HCT116 cells overexpressing Vec, PRL-3 or C104S were cultured for 1 h in full media under normoxia (Ctrl) or amino-acid starved (AA-) conditions, prior to analysis as in (a). (c) HCT116 cells stably expressing shRNA against PRL-3 (shPRL-3) or control shRNA (shCon) were cultured and analysed as in (a). (d) Cell lysates from (a) were immunoprecipitated with a configuration-specific anti-Rheb-GTP antibody and analysed by immunoblotting with the indicated antibodies. (e) Cell lysates from (b) were analysed as in (d). (f) Proposed signalling pathway for PRL-3 to mTOR substrates, 4EBP1 and p70S6K. Representative full immunoblots for the cropped images presented here are provided in the Supplementary Information.