Regulation of intracellular mTORC2 activity by PI3K. (A and B) Intracellular membrane compartments display distinct mTORC2 sensitivity to PI3K inhibition. HEK293T cells were cotransfected with LocaTOR2 and the corresponding recruiter constructs for 18 h, serum starved overnight, and then preincubated for 30 min with 500 nM GDC-0941 or vehicle and treated with 250 nM AP21967 for 40 min. The cells were lysed, and the level of LocaTOR2 pSer474 phosphorylation was analyzed by quantitative WBs and normalized against total Akt. The data are presented as fold increase over untreated vehicle control averaged over several independent experiments (shown in parentheses); shown are means ± SEM. *, P < 0.05 (one-sample t test with Benjamin-Hochberg correction). EE, early endosome; LE, late endosome; PM, plasma membrane; mito, outer mitochondrial membrane; N, nontransfected control; RE, recycling endosome. (C) mTORC2 activity at the plasma membrane is insensitive to PI3K activity. HEK293T cells were cotransfected with LocaTOR2 and the plasma membrane recruiter construct for 18 h, grown with or without 10% serum (FBS) overnight, and then preincubated for 30 min with 500 nM GDC-0941 or the vehicle and treated with 250 nM AP21967 for 40 min. The cells were lysed, and the level of LocaTOR2^pSer474 was determined by quantitative WBs. The results are plotted as the fold increase over vehicle control. The numbers in parentheses indicate independent experiments; error bars are SEM. n.s., not significant. One-way analysis of variance: P > 0.5. (D) mTORC2 activity at the plasma membrane is insensitive to growth factors. HEK293T cells were cotransfected with LocaTOR2 and the plasma membrane recruiter construct for 18 h, serum starved overnight, pretreated for 40 min with 250 nM AP21967, and then stimulated by 100 ng/ml insulin. The cells were lysed, and pSer473 phosphorylation of the endogenous Akt and LocaTOR2 was determined using PAGE/WBs. ctrl, control; endo, endogenously expressed Akt.