The major target of the endogenously generated reactive oxygen species in response to insulin stimulation is phosphatase and tensin homolog and not phosphoinositide-3 kinase (PI-3 kinase) in the PI-3 kinase/Akt pathway - PubMed (original) (raw)

The major target of the endogenously generated reactive oxygen species in response to insulin stimulation is phosphatase and tensin homolog and not phosphoinositide-3 kinase (PI-3 kinase) in the PI-3 kinase/Akt pathway

Ji Hae Seo et al. Mol Biol Cell. 2005 Jan.

Abstract

Phosphoinositide-3 kinase (PI-3 kinase) and its downstream signaling molecules PDK-1 and Akt were analyzed in SK-N-SH and SK-N-BE(2) human neuroblastoma cell lines. When cells were stimulated with insulin, PI-3 kinase was activated in both cell lines, whereas the translocation of PDK-1 to the membrane fraction and phosphorylated Akt were observed only in SK-N-SH cells. Analyses of the insulin-mediated reactive oxygen species (ROS) generation and Phosphatase and Tensin homolog (PTEN) oxidation indicate that PTEN oxidation occurred in SK-N-SH cells, which can produce ROS, but not in SK-N-BE(2) cells, which cannot increase ROS in response to insulin stimulation. When SK-N-SH cells were pretreated with the NADPH oxidase inhibitor diphenyleneiodonium chloride before insulin stimulation, insulin-mediated translocation of PDK-1 to the membrane fraction and phosphorylation of Akt were remarkably reduced, whereas PI-3 kinase activity was not changed significantly. These results indicate that not only PI-3 kinase activation but also inhibition of PTEN by ROS is needed to increase cellular level of phosphatidylinositol 3,4,5-trisphosphate for recruiting downstream signaling molecules such as PDK-1 and Akt in insulin-mediated signaling. Moreover, the ROS generated by insulin stimulation mainly contributes to the inactivation of PTEN and not to the activation of PI-3 kinase in the PI-3 kinase/Akt pathway.

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Figures

Figure 1.

Insulin stimulation increases PI-3 kinase activity in SKN-SH and SK-N-BE(2) cells. Cells treated with insulin (100 nM) for the indicated time, were lysed and immunoprecipitated with either anti-phosphotyrosine (PY20, 5 μg) (A) or anti-p85 (B). Kinase activity was measured as described under Materials and Methods by using phosphatidyl inositol as substrate, and the product PIP was resolved by TLC. Incorporation of 32P into PIP was imaged and quantitated using a PhosphorImager. Each bar represents the quantitated average ± SE for three independent experiments normalized to the value of unstimulated SK-N-SH cells. The anti-p85 blot (B) was obtained from the protein A beads used for PI-3 kinase activity assay.

Figure 2.

Effect of insulin or H2O2 on the PDK-1 recruitment to the membrane fraction (A) and Akt activation (B and C) in SK-N-SH and SK-N-BE(2) cells. Membrane fractions (A) or whole cell lysates (B and C) (50 μg of protein) prepared from cells treated with insulin (100 nM), H2O2 (1 mM), or insulin plus H2O2 for 10 min were analyzed with anti-PDK-1 (A), or anti-phospho-Akt (Ser473) (B and C). The same blots were reprobed with anti-actin (A) and anti-Akt (B and C), respectively, to confirm equal protein loading.

Figure 3.

Effect of insulin on ROS generation in SK-N-SH and SK-N-BE(2)C cells. The generation of ROS was assayed on the basis of DCF fluorescence as described in Materials and Methods. Confocal micrographs (A) of DCF fluorescence from the SK-N-SH and SK-NBE(2) cells either unstimulated or stimulated with insulin (100 nM) for 15 min. Time course of the ROS generation by insulin in SKN-SH (black bar) and SK-N-BE(2) (gray bar) cells. The relative fluorescence value of each time point was normalized to the value of unstimulated SK-N-SH cells. Each bar represents the average ± SE of three independent experiments.

Figure 4.

Oxidation of PTEN and recovery of PTEN activity from oxidative inactivated PTEN in the cells stimulated either insulin or H2O2. The oxidation of PTEN was analyzed in SK-N-SH cells stimulated with insulin (A) or treated with DPI (10 μM) for 30 min before insulin stimulation (B) and in SK-N-BE(2) cells stimulated with insulin (C) or H2O2 (D). SK-N-SH and SK-N-BE(2) cells were stimulated for the indicated times with insulin (100 nM), DPI (10 μM) plus insulin, or H2O2 (1 mM) and processed by the procedure described in Materials and Methods. Proteins, 200 μg (A and B), 400 μg (C), or 50 μg (D), were fractionated on a 10% nonreducing SDS-PAGE. The redox state of PTEN was analyzed using anti-PTEN antibody immunoblotting to identify shifts in PTEN mobility. The recovery of PTEN phosphatase activity from oxidative inactivated PTEN was measured in anti-PTEN immunoprecipitates from the cells stimulated with insulin or H2O2 for 15 min (E). Free cysteine residues of PTEN were blocked with irreversible alkylating reagent NEM immediately after cells were stimulated with insulin or H2O2 as described in Materials and Methods. The oxidized fraction of PTEN in anti-PTEN immunoprecipitates was converted to active form by reducing with DTT. The phosphatase activity was measured using 32P-labeled PtdIns(3,4,5)P3 as substrates. Each bar represents the average ± SE of the two independent experiments.

Figure 5.

Analyses of the phosphorylation status of PTEN. Cells were stimulated with insulin (100 nM) or H2O2 (1 mM) for 10 min, lysed, and immunoprecipitated using anti-PTEN (5 μg) as described in Materials and Methods. The anti-PTEN immunoprecipitates were fractionated on a 10% SDS-PAGE, transferred to nitrocellulose, and blotted with anti-phosphotyrosine (4G10) (A), anti-phospho-PTEN (B), and anti-PTEN.

Figure 6.

PI-3 kinase/Akt pathway regulation. (A) Effect of the NADPH oxidase inhibitor DPI and exogenous H2O2 on PI-3 kinase/Akt pathway. The PI-3 kinase activity was measured in anti-phosphotyrosine immunoprecipitates from cells treated for 10 min with insulin, H2O2, insulin plus DPI, or insulin plus H2O2. Each bar represents the mean value ± SE of three independent experiments normalized to the value of unstimulated SK-N-SH cells. (B) Effect of DPI and LY294002 on the insulin-mediated translocation of PDK-1 and Akt activation in SK-N-SH cells. Membrane fractions and lysates were prepared from cells stimulated with insulin, insulin plus DPI, or insulin plus LY294002. Samples were fractionated (50 μg of protein) by 10% SDS-PAGE and transferred to nitrocellulose. PDK-1 and activated Akt were visualized using anti-PDK-1 and anti-phospho-Akt. The same blots were reprobed with anti-actin and anti-Akt, respectively, to confirm equal protein loading. (C) Effect of PI-3 kinase inhibitor on the Akt activation in response to exogenous H2O2. Proteins (50 μg) from cells stimulated with H2O2 or H2O2 plus LY294002, were fractionated on two 10% SDS-PAGs and transferred to nitrocellulose. One blot was used for visualizing phosphorylated Akt by using anti-phospho-Akt, and the other blot was used for visualizing Akt by using anti-Akt to confirm equal protein loading.

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The major target of the endogenously generated reactive oxygen species in response to insulin stimulation is phosphatase and tensin homolog and not phosphoinositide-3 kinase (PI-3 kinase) in the PI-3 kinase/Akt pathway - PubMed (original) (raw)