Figure 2.

Alkaline extracellular pH is sufficient to increase mTORC2 catalytic activity and signaling.A, MEFs in complete media (DMEM/FBS) were refed with the same media at either pH 7.4 or 8.3 for various times (5–60 min). Whole-cell lysates (WCLs) were immunoblotted as indicated. B, similar to panel A, except cells were pretreated with Torin 1 (T). Graph, mean ratio ± SD of Akt P-S473/Akt; n = 4 independent experiments. ∗∗p < 0.01 using one-way ANOVA and Tukey’s post hoc tests. C, MEFs in complete media (DMEM/FBS) were pretreated with Torin 1 (T) and stimulated without (−) or with (+) NH₄Cl (5–10 min). D, similar to panel A, except cells were pretreated with cariporide (Cari) (30 min). E, Rictor was immunoprecipitated (IP) from MEFs that had been serum-starved, pretreated with Torin 1 (T), and refed with serum-free DMEM at pH 7.4 or 8.3 (10 min) −/+ Torin 1. In vitro kinase (IVK) reactions were performed with ATP and His-Akt1 substrate, with Torin 1 present in the IVK reaction (lane 3). IVKs and WCLs were immunoblotted as indicated. F, Rictor^−/− MEFs rescued with either vector control (V) or HA-Rictor were treated as in panel A. FBS, fetal bovine serum; MEFs, mouse embryonic fibroblasts; mTOR, mechanistic target of rapamycin; mTORC2, mTOR complex 2; NH₄Cl, ammonium chloride.