Fig. 1.

Akt suppression of autophagy, interaction with Beclin 1, and phosphorylation of Beclin 1. (A) Biochemical assessment of autophagy (p62 and LC3) and mTOR activity (p-4E-BP1) in HeLa cells expressing constitutively active Akt (myr-Akt1) or control vector, grown in normal medium or starved in Earles’ Balance Salt Solution (EBSS) for 2 hours, and treated with 250 nM Torin 1 or control solution. (B) GFP-LC3 dots (autophagosomes) in HeLa/GFP-LC3 cells treated as in (A). Bars represent mean ± SEM of triplicate samples with >50 cells analyzed per sample. Similar results observed in 3 independent experiments. (C) Immunoprecipitation of endogenous Akt with endogenous Beclin 1 in HeLa cells with or without starvation for 2 hours. (D) In vitro phosphorylation of Flag-Beclin 1 S²⁹⁵ by GST-Akt1 with or without 1 μM indicated Akt inhibitors. (E) Phosphorylation of endogenous Beclin 1 S²⁹⁵ and Beclin 1 S²³⁴ in HeLa cells transfected with control vector or HA-myr-Akt1 with or without Torin1 for 4 hours. (E) Phosphorylation of endogenous Beclin 1 S²⁹⁵ in paired melanoma (WM793 and 451Lu), glioblastoma (U87-MG, U87-MG + PTEN), and breast cancer (MCF-10DCIS and MDMB-231) cells with high and low activities of Akt, respectively. *P<0.05; **P<0.01; Tukey test. WCL, whole cell lysates.