Figure 5: The p110α–PDK1–RSK2 signalling axis regulates ATF4 protein stability.

(a,b) ATF4 ubiquitination levels are higher in a HCT116 WT clone than the PIK3CA mutant clone. HCT116 PIK3CA WT and mutant cells were treated with 5 μM MG132 for 6 h. Endogenous ATF4 proteins were immunoprecipitated and blotted with an anti-ubiquitin antibody (a). FLAG-tagged ATF4 and human influenza hemagglutinin (HA)-tagged ubiquitin plasmids were co-transfected into HCT116 PIK3CA WT and mutant cells. FLAG-tagged ATF4 was immunoprecipitated and blotted with anti-HA antibody to detect HA-tagged ubiquitin (b). (c,d) Inhibitors of PI3K, PDK1 and RSK2 reduce ATF4 protein levels in a HCT116 PIK3CA mutant clone. Inhibitors include 10 μM LY294002 (pan-PI3K inhibitor), 1 or 5 μM BYL-719 (p110α-specific inhibitor), 5 μM BEZ235 (PI3K/mTOR dual-specificity inhibitor), 6 μM GSK2334470 (PDK1 inhibitor), 10 μM GSK690693 (AKT inhibitor), 10 μM CHIR-99021 (GSK3β inhibitor), 10 μM rapamycin (mTOR inhibitor), 10 μM BI-D1870 (pan-RSK inhibitor) and 10 μM FMK (RSK2 inhibitor). Cells were treated with vehicle or each inhibitor for 12 h and lysates were blotted with the indicated antibodies. (e) A schematic of the p110α signalling pathway that regulates ATF4 protein stability. (f) Overexpression of oncogenic p110α mutants increases ATF4 and GPT2 protein levels. The indicated constructs were transfected into HCT116 WT cells. Cell lysates were blotted with the indicated antibodies. (g) A kinase-dead mutation combined with the p110α E545K mutation in the same allele reduces protein levels of ATF4 and GPT2. A PIK3CA D993A kinase-dead mutant was knocked into a DLD1 PIK3CA mutant clone as described in Supplementary Fig. 5e,f. Cell lysates were blotted with the indicated antibodies. (h) Genetic inactivation of p110α enzymatic activity renders DLD1 PIK3CA oncogenic mutant cells less sensitive to glutamine deprivation. Clones of the indicated genotypes were grown with or without glutamine for 72 h. Apoptotic cells were quantified. Data presented as mean±s.e.m. of three independent cultures. **P<0.01, t-test. (i) Knockdown of PDK1 by two independent siRNAs reduces ATF4 and GPT2 protein levels in both HCT116 and DLD1 PIK3CA mutant clones. (j) Knockdown of RSK2 by two independent siRNAs reduces ATF4 and GPT2 protein levels.