Akt-mediated regulation of autophagy and tumorigenesis through Beclin 1 phosphorylation - PubMed (original) (raw)

Akt-mediated regulation of autophagy and tumorigenesis through Beclin 1 phosphorylation

Richard C Wang et al. Science. 2012.

Abstract

Aberrant signaling through the class I phosphatidylinositol 3-kinase (PI3K)-Akt axis is frequent in human cancer. Here, we show that Beclin 1, an essential autophagy and tumor suppressor protein, is a target of the protein kinase Akt. Expression of a Beclin 1 mutant resistant to Akt-mediated phosphorylation increased autophagy, reduced anchorage-independent growth, and inhibited Akt-driven tumorigenesis. Akt-mediated phosphorylation of Beclin 1 enhanced its interactions with 14-3-3 and vimentin intermediate filament proteins, and vimentin depletion increased autophagy and inhibited Akt-driven transformation. Thus, Akt-mediated phosphorylation of Beclin 1 functions in autophagy inhibition, oncogenesis, and the formation of an autophagy-inhibitory Beclin 1/14-3-3/vimentin intermediate filament complex. These findings have broad implications for understanding the role of Akt signaling and intermediate filament proteins in autophagy and cancer.

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Figures

Fig. 1

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.

Fig. 2

Fig. 2

Effect of a non-phosphorylatable mutant of Beclin 1 (Beclin 1 S234A/S295A (AA)) on autophagy, anchorage-independent growth, and Akt-mediated tumorigenesis. (A) Biochemical assessment of autophagy in Rat2 fibroblasts expressing indicated Flag-Beclin 1 and Akt constructs. (B) GFP-LC3 dots in Rat2 cells transduced with indicated lentiviral vectors and transfected with GFP-LC3. Bars represent mean ± SEM of triplicate samples with >50 cells analyzed per sample. Similar results observed in 3 independent experiments. (C) Soft agar colonies formed by Rat2 fibroblasts transduced with indicated vectors. Bars represent mean ± SEM of triplicate samples of ≥12 random images analyzed by ImageJ. Similar results observed in 3 independent experiments. (D) Xenograft tumor volumes formed by Rat2 fibroblasts transduced with indicated vectors. (P<0.001 for myr-Akt1 + Beclin 1 AA group vs. myr-Akt1 + vector or myr-Akt1 + Beclin 1 groups; linear-mixed effect model). (E) Tumor weights at day 21. (F) Representative images of tumors formed by Rat2 fibroblasts. Arrows denote representative p62-, Ki-67-, and TUNEL-positive cells. Scale bars, 20 μm. For (D) and (E), results represent mean ± SEM for all tumors in each genotype (9–10 per group). *P<0.05, **P<0.01, ***P<0.001; Tukey-test.

Fig. 3

Fig. 3

Interactions between Beclin 1 and 14-3-3 proteins or Beclin 1 and vimentin promoted by active Akt. (A) Immunoprecipitation of 14-3-3 proteins with endogenous Beclin 1 in HeLa cells with or without starvation for 2 hours. (B to C) Endogenous Beclin 1 S295 phosphorylation (B) and immunoprecipitation of endogenous 14-3-3 and vimentin with endogenous Beclin 1 (C) in Rat2 fibroblasts tranduced with control vector or myr-Akt1. (D) Immunoprecipitation of endogenous 14-3-3 and vimentin with Flag-Beclin 1 in Rat2 cells transduced with indicated Flag-Beclin 1 and Akt constructs. *, non-specific band. (E) Localization of Flag-Beclin 1 with endogenous vimentin in Rat2 cells transduced with the indicated vectors. WCL, whole cell lysates.

Fig. 4

Fig. 4

Effects of vimentin on autophagy and Akt-mediated transformation. (A) GFP-LC3 dots in Rat2 cells transduced with indicated vimentin shRNA and transfected with GFP-LC3. Bars represent mean ± SEM of triplicate samples with >50 cells analyzed per sample. Similar results observed in 3 independent experiments. (B) Biochemical assessment of autophagy in Rat2 cells transduced with indicated vimentin shRNA and myr-Akt1 or control vector. (C) Soft agar colonies formed by Rat2 fibroblasts transduced with indicated vectors. Bars represent mean ± SEM of triplicate samples of ≥12 random images analyzed by ImageJ. (D) Speculative model for relations between Akt phosphorylation of Beclin 1; formation of a complex with Beclin 1, 14-3-3 proteins, and intermediate filament (IF) proteins; autophagy, and tumorigenesis. *P<0.05, **P<0.01, ***P<0.001; t test. In (A) and (C), all comparisons are with the first bar in graph.

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