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Science 338 (6109): 956-959

Copyright © 2012 by the American Association for the Advancement of Science

Akt-Mediated Regulation of Autophagy and Tumorigenesis Through Beclin 1 Phosphorylation

Richard C. Wang1,2, Yongjie Wei2,3,4, Zhenyi An2,3, Zhongju Zou2,3,4, Guanghua Xiao5, Govind Bhagat6, Michael White7, Julia Reichelt8, and Beth Levine2,3,4,9,*

1 Department of Dermatology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
2 Center for Autophagy Research, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
3 Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
4 Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
5 Department of Clinical Sciences, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
6 Department of Pathology and Cell Biology, Columbia University Medical Center and New York Presbyterian Hospital, New York, NY 10032, USA.
7 Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
8 Institute of Cellular Medicine, University of Newcastle, Framlington Place, NE2 4HH Newcastle upon Tyne, UK.
9 Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.


Figure 1
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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 Torin1 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 three 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 hemagglutinin–myr-Akt1 with or without Torin1 for 4 hours. (F) 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 < 0.01, ***P < 0.001; Tukey test. WCL, whole-cell lysates.

 

Figure 2
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Fig. 2. Effect of a nonphosphorylatable 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 three 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 using ImageJ. Similar results observed in three independent experiments. (D) Xenograft tumor volumes formed by Rat2 fibroblasts transduced with indicated vectors (P < 0.001 for myr-Akt1 + Beclin 1 AA group versus 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 or 10 per group). *P < 0.05, **P < 0.01, ***P < 0.001; Tukey test.

 

Figure 3
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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 and 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. Asterisk indicates nonspecific band. (E) Localization of Flag-Beclin 1 with endogenous vimentin in Rat2 cells transduced with the indicated vectors. WCL, whole-cell lysates.

 

Figure 4
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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 three 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 using 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; Student’s t test. In (A) and (C), all comparisons are with the first bar in graph.

 


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