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. 2009 May;91(5):577-85.
doi: 10.1016/j.biochi.2009.01.010. Epub 2009 Feb 6.

Hydrogen peroxide-induced Akt phosphorylation regulates Bax activation

Mahdieh Sadidi  1 , Stephen I Lentz, Eva L Feldman
Affiliations

Hydrogen peroxide-induced Akt phosphorylation regulates Bax activation

Mahdieh Sadidi et al. Biochimie. 2009 May.

Abstract

Reactive oxygen species such as hydrogen peroxide (H(2)O(2)) are involved in many cellular processes that positively and negatively regulate cell fate. H(2)O(2), acting as an intracellular messenger, activates phosphatidylinositol-3 kinase (PI3K) and its downstream target Akt, and promotes cell survival. The aim of the current study was to understand the mechanism by which PI3K/Akt signaling promotes survival in SH-SY5Y neuroblastoma cells. We demonstrate that PI3K/Akt mediates phosphorylation of the pro-apoptotic Bcl-2 family member Bax. This phosphorylation suppresses apoptosis and promotes cell survival. Increased survival in the presence of H(2)O(2) was blocked by LY294002, an inhibitor of PI3K activation. LY294002 prevented Bax phosphorylation and resulted in Bax translocation to the mitochondria, cytochrome c release, caspase-3 activation, and cell death. Collectively, these findings reveal a mechanism by which H(2)O(2)-induced activation of PI3K/Akt influences post-translational modification of Bax and inactivates a key component of the cell death machinery.

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Figures

Fig. 1
Fig. 1
H2O2 stimulates phosphorylation of Akt in SH-SY5Y Cells. (A) Serum starved SH-SY5Y cells were treated with 0.1 mM H2O2 for the indicated times. Whole cell lysates were analyzed by Western blotting, using an anti-phospho-Akt antibody (1:1000, upper panel). Blots were then stripped and blotted for Akt protein (1:1000, lower panel). Cells were pre-treated with or without 20 μM LY294002 for 1 h, followed by H2O2 treatment for 4 h (B) or for 24 h (C) and immunoblotted for phospho-Akt as indicated (upper panels). Blots were then stripped and blotted for Akt protein (lower panels). Data are from one of three representative experiments.
Fig. 2
Fig. 2
Effect of H2O2 on cell death. Serum deprived SH-SY5Y cells with no treatment for 24 h (control), and treated with 0.1 mM H2O2 for 24 h without LY pre-treatment (H2O2) or with pre-treatment with 20 μM LY294002 (LY+H2O2). DNA was stained with propidium iodide and DNA content was measured by flow cytometry. Percent cell death is shown as mean ± S.E.M. for four separate experiments. Treatment of the serum-starved cells with H2O2 alone was significantly different from control (*, p < 0.05). Serum-starved cells with pre-treatment with LY and then H2O2 addition (LY+H2O2) were significantly different from both control and from H2O2 alone (*, p < 0.01).
Fig. 3
Fig. 3
Bax phosphorylation in SH-SY5Y Cells. (A) Cells were treated with H2O2 for the indicated times. Cell lysates were immunoprecipitated with anti-Bax antibody (I.P.Bax) and then immunoblotted with Bax antibody. (A′) Whole cell lysates were analyzed by Western blotting, using Bax antibody. (B) Cells were treated with H2O2 for the indicated times. Cell lysates were immunoprecipitated with anti-Bax antibody (I.P.Bax) and then immunoblotted with anti-phosphoserine antibody. (B′) Cells were pre-treated with or without LY, and then treated with H2O2 for 4 h. Cell lysates were immunoprecipitated with Bax antibody and then immunoblotted with anti- phosphoserine antibody. (C) 24 h time course; Cell lysates from control (no treatment), H2O2 alone, LY + H2O2, and H2O2 + PP-2A/C were immunoprecipitated with anti- phosphoserine antibody, and then immunoblotted with Bax antibody. (D) Cells were pre-treated with or without Wortmannin (W), and then treated with H2O2 for24 h. Cell lysates were immunoprecipitated with anti- phosphoserine antibody and then immunoblotted with Bax antibody.
Fig. 4
Fig. 4
Bax translocation to the mitochondria following PI3K inhibition. (A–C) Control SH-SY5Y cells; (D–F) cells treated with 0.1 mM H2O2 for 24 h; (G–I) cells pre-treated with PI3K inhibitor (20 μM of LY294002, LY) and then treated with 0.1 mM H2O2 for 24 h (LY + H2O2). Cells were double immunostained with Bax (N-20, red), Cox1 (green) and nuclei were visualized by DAPI (blue). Asterisks indicate cells in inserts. Normal healthy control cells are shown in A and C. In H2O2 only treatments (D–F), cells are healthy with intact nuclei and diffuse Bax staining in the cytoplasm. PI3K inhibition resulted in punctate Bax staining (G) and Bax translocation to mitochondria (H) as is evident by yellow signal in the merged image (I). Bars = 10 μm.
Fig. 5
Fig. 5
Cytochrome c release into the cytoplasm following PI3K inhibition. (A, B) Control cells; (C, D) cells treated with 0.1 mM H2O2 for 24 h; (E, F) cells pre-treated with 20 μM of LY294002 (LY), followed by 0.1 mM H2O2 treatment for 24 h (LY + H2O2). Cells were immunostained with cytochrome c (green) and nuclei were visualized by DAPI staining (blue). Asterisks indicate cells in inserts. Healthy cells (A, B and C, D) show punctate cytochrome c staining. Apoptotic cells with diffused cytochrome c (E), and condensed nuclei (F) are shown by red arrowheads. Bars = 10μm.
Fig. 6
Fig. 6
Inhibition of PI3K pathway lead to caspase-3 activation. (A–C) Control; (D–F) SH-SY5Y cells treated with 0.1 mM H2O2 for 24 h; (G–I) 20 μM of LY294002 (LY) followed by 0.1 mM H2O2 for 24 h (LY + H2O2). Cells were immunostained with cleaved caspase-3 (red), and nuclei were visualized by DAPI staining (blue). Caspase-3 activation is shown by the presence of red staining in G. Red arrowheads indicate cleaved caspase-3 staining in cells with condensed nuclei (I). Merged fluorescence signals are overlaid onto phase images (C, F, I) Bars = 10μm.
Fig. 7
Fig. 7
Activation of Akt by H2O2 regulates the activity of the pro-apoptotic Protein Bax. H2O2, that produces a mild oxidative environment, activates the PI3K/Akt survival pathway (blue arrows). When Akt is activated by H2O2, Bax is located in the cytoplasm in its phosphorylated (inactive) form and the cell survives (blue arrows). Inhibition of the PI3K pathway results in dephosphorylation of Bax and its translocation to the mitochondria (red arrows). Insertion of Bax into the mitochondria leads to cytochrome c release (possibly through Bax pore formation), caspase-3 activation, and apoptosis (red arrows). Abbreviations: MOM, mitochondrial outer membrane; MIM: mitochondrial inner membrane; MIS, mitochondrial intermembrane space.
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