PH domain targets mSin1/mTORC2 to cellular membranes. (A) Schematic representation of mSin1 isoforms 1, 2, and 5. Yellow boxes represent the PH domain. (B) Isoforms 1, 2, and 5 of mSin1-GFP are incorporated into mTORC2. HEK293T cells were transiently transfected with GFP or the corresponding mSin1-GFP isoform, and lysed, and GFP-tagged proteins were pulled down using GFP-Trap beads. Shown is a representative pull-down result. n = 2 for mSin1.1 and mSin1.5; n = 3 for mSin1.2. (C) In vitro kinase assay with immunoprecipitated mTORC2. HEK293T cells transiently transfected with GFP or mSin1.2^WT-GFP were stimulated with 100 ng/ml insulin for 10 min and lysed, and GFP-tagged proteins were pulled down using GFP-Trap beads. The beads were preincubated with or without 100 nM Torin1 and incubated for 1 h at 37°C with purified dephosphorylated recombinant full-length human Akt1 with or without 0.5 mM ATP, and pSer473 phosphorylation was determined using PAGE/WBs. Shown is a representative result of two independent experiments. (D) Intracellular localization of isoforms 1, 2, and 5 of mSin1-GFP in HeLa cells. (E) mSin1 localizes to the plasma membrane and endosomal vesicles. HeLa cells were transiently cotransfected with mSin1.2-GFP and markers of the plasma membrane (PM; mCherry-KRas4B^C30) along with early (mCherry-Rab5) and late endosomes (mCherry-Rab7). (F) The PH domain targets mSin1 to cellular membranes. HeLa cells were transiently transfected with mSin1.2^WT-GFP or mSin1ΔPH-GFP. (D–F) Bars, 10 µm. (G) The PH domain is dispensable for mSin1 incorporation into mTORC2. HeLa cells were transiently transfected with mSin1.2^WT-GFP or mSin1.2^ΔPH-GFP and lysed, and GFP-tagged proteins were pulled down using GFP-Trap beads. IP, immunoprecipitate. (H) Full-length mSin1 does not change localization upon growth factor treatment or PI3K inhibition. MCF7 cells cotransfected with mSin1.2^WT-GFP and PH^Akt-mCherry for 18 h were starved overnight and then treated first with 100 ng/ml insulin and then with 500 nM GDC-0941. Shown are the time traces of mean fluorescence intensity in a cytosolic region of interest. Data are presented as means ± SEM. n = 2; 18 cells. Normalized to t = –150 s.